Antisense modulation of human MDM2 expression

ABSTRACT

Compounds, compositions and methods are provided for inhibiting the expression of human mdm2. The compositions include antisense compounds targeted to nucleic acids encoding mdm2. Methods of using these oligonucleotides for inhibition of mdm2 expression and for treatment of diseases such as cancers associated with overexpression of mdm2 are provided.

[0001] This application is a continuation of U.S. patent application No.09/280,805 filed Mar. 26, 1999 which is a continuation-in-part of U.S.patent application No. 09/048,810 filed Mar. 26, 1998.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for modulatingexpression of the human mdm2 gene, a naturally present cellular geneimplicated in abnormal cell proliferation and tumor formation. Thisinvention is also directed to methods for inhibiting hyperproliferationof cells; these methods can be used diagnostically or therapeutically.Furthermore, this invention is directed to treatment of conditionsassociated with expression of the human mdm2 gene.

BACKGROUND OF THE INVENTION

[0003] Inactivation of tumor suppressor genes leads to unregulated cellproliferation and is a cause of tumorigenesis. In many tumors, the tumorsuppressors, p53 or Rb (retinoblastoma) are inactivated. This can occureither by mutations within these genes, or by overexpression of the mdm2gene. The mdm2 protein physically associates with both p53 and Rb,inhibiting their function. The levels of mdm2 are maintained through afeedback loop mechanism with p53. Overexpression of mdm2 effectivelyinactivates p53 and promotes cell proliferation.

[0004] The role of p53 in apoptosis and tumorigenesis is well-known inthe art (see, in general, Canman, C. E. and Kastan, M. B., Adv.Pharmacol., 1997, 41, 429-460). Mdm2 has been shown to regulate p53'sapoptotic functions (Chen, J., et al., Mol. Cell Biol., 1996, 16,2445-2452; Haupt, Y., et al., EMBO J., 1996, 15, 1596-1606).Overexpression of mdm2 protects tumor cells from p53-mediated apoptosis.Thus, mdm2 is an attractive target for cancers associated with alteredp53 expression.

[0005] Amplification of the mdm2 gene is found in many human cancers,including soft tissue sarcomas, astrocytomas, glioblastomas, breastcancers and non-small cell lung carcinomas. In many blood cancers,overexpression of mdm2 can occur with a normal copy number. This hasbeen attributed to enhanced translation of mdm2 mRNA, which is thoughtto be related to a distinct 5′-untranslated region (5′-UTR) which causesthe transcript to be translated more efficiently than the normal mdm2transcript. Landers et al., Cancer Res. 57, 3562, (1997).

[0006] Several approaches have been used to disrupt the interactionbetween p53 and mdm2. Small peptide inhibitors, screened from a phagedisplay library, have been shown in ELISA assays to disrupt thisinteraction [Bottger et al., J. Mol. Biol., 269, 744 (1997)].Microinjection of an anti-mdm2 antibody targeted to the p53-bindingdomain of mdm2 increased p53-dependent transcription [Blaydes et al.,Oncogene, 14, 1859 (1997)].

[0007] A vector-based antisense approach has been used to study thefunction of mdm2. Using a rhabdomyosarcoma model, Fiddler et al. [Mol.Cell Biol., 16, 5048 (1996)] demonstrated that amplified mdm2 inhibitsthe ability of MyoD to function as a transcription factor. Furthermore,expression of full-length antisense mdm2 from a cytomegaloviruspromoter-containing vector restores muscle-specific gene expression.

[0008] Antisense oligonucleotides have also been useful in understandingthe role of mdm2 in regulation of p53. An antisense oligonucleotidedirected to the mdm2 start codon allowed cisplatin-induced p53-mediatedapoptosis to occur in a cell line overexpressing mdm2 [Kondo et al.,Oncogene, 10, 2001 (1995)]. The same oligonucleotide was found toinhibit the expression of P-glycoprotein [Kondo et al., Br. J. Cancer,74, 1263 (1996)]. P-glycoprotein was shown to be induced by mdm2. Teohet al [Blood, 90, 1982 (1997)] demonstrated that treatment with anidentical mdm2 antisense oligonucleotide or a shorter version within thesame region in a tumor cell line decreased DNA synthesis and cellviability and triggered apoptosis.

[0009] Chen et al. [Proc. Natl. Acad. Sci. USA, 95, 195 (1998); WO99/10486] disclose antisense oligonucleotides targeted to the codingregion of mdm2. A reduction in mdm2 RNA and protein levels was seen, andtranscriptional activity from a p53-responsive promoter was increasedafter oligonucleotide treatment of JAR (choriocarcinoma) or SJSA(osteosarcoma) cells.

[0010] WO 93/20238 and WO 97/09343 disclose, in general, the use ofantisense constructs, antisense oligonucleotides, ribozymes andtriplex-forming oligonucleotides to detect or to inhibit expression ofmdm2. EP 635068B1, issued Nov. 5, 1997, describes methods of treating invitro neoplastic cells with an inhibitor of mdm2, and inhibitorycompounds, including antisense oligonucleotides and triple-strandforming oligonucleotides.

[0011] There remains a long-felt need for improved compositions andmethods for inhibiting mdm2 gene expression.

SUMMARY OF THE INVENTION

[0012] The present invention provides antisense compounds which aretargeted to nucleic acids encoding human mdm2 and are capable ofmodulating, and preferably, inhibiting mdm2 expression. The presentinvention also provides chimeric compounds targeted to nucleic acidsencoding human mdm2. The antisense compounds of the invention arebelieved to be useful both diagnostically and therapeutically, and arebelieved to be particularly useful in the methods of the presentinvention.

[0013] The present invention also comprises methods of inhibiting theexpression of human mdm2, particularly the increased expressionresulting from amplification of mdm2. These methods are believed to beuseful both therapeutically and diagnostically as a consequence of theassociation between mdm2 expression and hyperproliferation. Thesemethods are also useful as tools, for example, for detecting anddetermining the role of mdm2 expression in various cell functions andphysiological processes and conditions and for diagnosing conditionsassociated with mdm2 expression.

[0014] The present invention also comprises methods of inhibitinghyperproliferation of cells using compounds of the invention. Thesemethods are believed to be useful, for example, in diagnosingmdm2-associated cell hyperproliferation. Methods of treating abnormalproliferative conditions associated with mdm2 are also provided. Thesemethods employ the antisense compounds of the invention. These methodsare believed to be useful both therapeutically and as clinical researchand diagnostic tools.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Tumors often result from genetic changes in cellular regulatorygenes. Among the most important of these are the tumor suppressor genes,of which p53 is the most widely studied. Approximately half of all humantumors have a mutation in the p53 gene. This mutation disrupts theability of the p53 protein to bind to DNA and act as a transcriptionfactor. Hyperproliferation of cells occurs as a result. Anothermechanism by which p53 can be inactivated is through overexpression ofmdm2, which regulates p53 activity in a feedback loop. The mdm2 proteinbinds to p53 in its DNA binding region, preventing its activity. Mdm2 isamplified in some human tumors, and this amplification is diagnostic ofneoplasia or the potential therefor. Over one third of human sarcomashave elevated mdm2 sequences. Elevated expression may also be involvedin other tumors including but not limited to those in which p53inactivation has been implicated. These include colorectal carcinoma,lung cancer and chronic myelogenous leukemia.

[0016] Many abnormal proliferative conditions, particularlyhyperproliferative conditions, are believed to be associated withincreased mdm2 expression and are, therefore believed to be responsiveto inhibition of mdm2 expression. Examples of these hyperproliferativeconditions are cancers, psoriasis, blood vessel stenosis (e.g.,restenosis or atherosclerosis), and fibrosis, e.g., of the lung orkidney. Increased levels of wild-type or mutated p53 have been found insome cancers (Nagashima, G., et al., Acta Neurochir. (Wein), 1999, 141,53- 61; Fiedler, A., et al., Langenbecks Arch. Surg., 1998, 383,269-275). Increased levels of p53 is also associated with resistance ofa cancer to a chemotherapeutic drug (Brown, R., et al., Int. J. Cancer,1993, 55, 678-684). These diseases or conditions may be amenable totreatment by induction of mdm2 expression.

[0017] The present invention employs antisense compounds, particularlyoligonucleotides, for use in modulating the function of nucleic acidmolecules encoding mdm2, ultimately modulating the amount of mdm2produced. This is accomplished by providing oligonucleotides whichspecifically hybridize with nucleic acids, preferably mRNA, encodingmdm2.

[0018] This relationship between an antisense compound such as anoligonucleotide and its complementary nucleic acid target, to which ithybridizes, is commonly referred to as “antisense”. “Targeting” anoligonucleotide to a chosen nucleic acid target, in the context of thisinvention, is a multistep process. The process usually begins withidentifying a nucleic acid sequence whose function is to be modulated.This may be, as examples, a cellular gene (or mRNA made from the gene)whose expression is associated with a particular disease state, or aforeign nucleic acid from an infectious agent. In the present invention,the target is a nucleic acid encoding mdm2; in other words, a mdm2 geneor RNA expressed from a mdm2 gene. mdm2 mRNA is presently the preferredtarget. The targeting process also includes determination of a site orsites within the nucleic acid sequence for the antisense interaction tooccur such that modulation of gene expression will result.

[0019] In accordance with this invention, persons of ordinary skill inthe art will understand that messenger RNA includes not only theinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form a region known to suchpersons as the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. Thus,oligonucleotides may be formulated in accordance with this inventionwhich are targeted wholly or in part to these associated ribonucleotidesas well as to the informational ribonucleotides. The oligonucleotide maytherefore be specifically hybridizable with a transcription initiationsite region, a translation initiation codon region, a 5′ cap region, anintron/exon junction, coding sequences, a translation termination codonregion or sequences in the 5′- or 3′-untranslated region. Since, as isknown in the art, the translation initiation codon is typically 5′-AUG(in transcribed mRNA molecules; 5′-ATG in the corresponding DNAmolecule), the translation initiation codon is also referred to as the“AUG codon,” the “start codon” or the “AUG start codon.” A minority ofgenes have a translation initiation codon having the RNA sequence5′-GUG, 5′UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shownto function in vivo. Thus, the terms “translation initiation codon” and“start codon” can encompass many codon sequences, even though theinitiator amino acid in each instance is typically methionine (ineukaryotes) or formylmethionine (prokaryotes). It is also known in theart that eukaryotic and prokaryotic genes may have two or morealternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAmolecule transcribed from a gene encoding mdm2, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region” and “translation initiation codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. This region is a preferred targetregion. Similarly, the terms “stop codon region” and “translationtermination codon region” refer to a portion of such an mRNA or genethat encompasses from about 25 to about 50 contiguous nucleotides ineither direction (i.e., 5′1 or 3′) from a translation termination codon.This region is a preferred target region. The open reading frame (ORF)or “coding region,” which is known in the art to refer to the regionbetween the translation initiation codon and the translation terminationcodon, is also a region which may be targeted effectively. Otherpreferred target regions include the 5′ untranslated region (5′UTR),known in the art to refer to the portion of an mRNA in the 5′ directionfrom the translation initiation codon, and thus including nucleotidesbetween the 5′ cap site and the translation initiation codon of an mRNAor corresponding nucleotides on the gene) and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA or corresponding nucleotides on the gene). mdm2 is believed to havealternative transcripts which differ in their 5′-UTR regions. The S-mdm2transcript class is translated approximately 8-fold more efficientlythan the L-mdm2 transcripts produced by the constitutive promoter.Landers et al., Cancer Res., 57, 3562 (1997). Accordingly, both the5′-UTR of the S-mdm transcript and the 5′-UTR of the L-mdm2 transcriptare preferred target regions, with the S-mdm2 5′-UTR being morepreferred. mRNA splice sites may also be preferred target regions, andare particularly useful in situations where aberrant splicing isimplicated in disease, or where an overproduction of a particular mRNAsplice product is implicated in disease. Aberrant fusion junctions dueto rearrangements or deletions may also be preferred targets.

[0020] Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired modulation.

[0021] “Hybridization”, in the context of this invention, means hydrogenbonding, also known as Watson-Crick base pairing, between complementarybases, usually on opposite nucleic acid strands or two regions of anucleic acid strand. Guanine and cytosine are examples of complementarybases which are known to form three hydrogen bonds between them. Adenineand thymine are examples of complementary bases which form two hydrogenbonds between them.

[0022] “Specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity such thatstable and specific binding occurs between the DNA or RNA target and theoligonucleotide.

[0023] It is understood that an oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment and, in the case of invitro assays, under conditions in which the assays are conducted.

[0024] Hybridization of antisense oligonucleotides with mRNA interfereswith one or more of the normal functions of mRNA. The functions of mRNAto be interfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.

[0025] The overall effect of interference with mRNA function ismodulation of mdm2 expression. In the context of this invention“modulation” means either inhibition or stimulation; i.e., either adecrease or increase in expression. This modulation can be measured inways which are routine in the art, for example by Northern blot assay ofmRNA expression as taught in the examples of the instant application orby Western blot or ELISA assay of protein expression, or by animmunoprecipitation assay of protein expression, as taught in theexamples of the instant application. Effects on cell proliferation ortumor cell growth can also be measured, as taught in the examples of theinstant application.

[0026] The antisense compounds of this invention can be used indiagnostics, therapeutics, prophylaxis, and as research reagents and inkits. Since these compounds hybridize to nucleic acids encoding mdm2,sandwich, calorimetric and other assays can easily be constructed toexploit this fact. Furthermore, since the antisense compounds of thisinvention hybridize specifically to nucleic acids encoding particularisozymes of mdm2, such assays can be devised for screening of cells andtissues for particular mdm2 isozymes. Such assays can be utilized fordiagnosis of diseases associated with various mdm2 forms. Provision ofmeans for detecting hybridization of oligonucleotide with a mdm2 gene ormRNA can routinely be accomplished. Such provision may include enzymeconjugation, radiolabelling or any other suitable detection systems.Kits for detecting the presence or absence of mdm2 may also be prepared.

[0027] The present invention is also suitable for diagnosing abnormalproliferative states in tissue or other samples from patients suspectedof having a hyperproliferative disease such as cancer or psoriasis. Theability of the oligonucleotides of the present invention to inhibit cellproliferation may be employed to diagnose such states. A number ofassays may be formulated employing the present invention, which assayswill commonly comprise contacting a tissue sample with an antisensecompound of the invention under conditions selected to permit detectionand, usually, quantitation of such inhibition. In the context of thisinvention, to “contact” tissues or cells with an antisense compoundmeans to add the compound(s), usually in a liquid carrier, to a cellsuspension or tissue sample, either in vitro or ex vivo, or toadminister the antisense compound(s) to cells or tissues within ananimal. Similarly, the present invention can be used to distinguishmdm2-associated tumors, particularly tumors associated with mdm2α, fromtumors having other etiologies, in order that an efficacious treatmentregime can be designed.

[0028] The antisense compounds of this invention may also be used forresearch purposes. Thus, the specific hybridization exhibited byoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

[0029] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleicacid. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent intersugar(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced binding to target and increased stability in thepresence of nucleases.

[0030] The antisense compounds in accordance with this inventionpreferably comprise from about 5 to about 50 nucleobases. Particularlypreferred are antisense oligonucleotides comprising from about 8 toabout 30 linked nucleobases (i.e. from about 8 to about 30 nucleosides).As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0031] Specific examples of some preferred modified oligonucleotidesenvisioned for this invention include those containingphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioates (usually abbreviated in the art as P═S) and thosewith CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂ [known as a methylene(methylimino)or MMI backbone], CH₂ —O—N(CH₃)—CH₂, CH₂— N(CH₃)—N(CH₃)—CH₂ andO—N(CH₃)—CH₂—CH₂ backbones, wherein the native phosphodiester (usuallyabbreviated in the art as P═O) backbone is represented as O—P—O—CH₂).Also preferred are oligonucleotides having morpholino backbonestructures (Summerton and Weller, U.S. Pat. No. 5,034,506). Furtherpreferred are oligonucleotides with NR—C(*)—CH₂—CH₂, CH₂—NR— C(*) —CH₂,CH₂—CH₂—NR—C(*), C(*) —NR—CH₂—CH₂ and CH₂—C(*) —NR—CH₂ backbones,wherein “*” represents O or S (known as amide backbones; DeMesmaeker etal., WO 92/20823, published Nov. 26, 1992). In other preferredembodiments, such as the peptide nucleic acid (PNA) backbone, thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleobases being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone (Nielsen et al.,Science, 254, 1497 (1991); U.S. Pat. No. 5,539,082). Other preferredmodified oligonucleotides may contain one or more substituted sugarmoieties comprising one of the following at the 2′ position: OH, SH,SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ orO(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN;CF₃; OCF₃; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a reporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. A preferred modificationincludes 2′-O-methoxyethyl [which can be written as 2′—O—CH₂CH₂OCH₃, andis also known in the art as 2′—O—(2-methoxyethyl) or 2′-methoxyethoxy][Martin et al., Helv. Chim. Acta, 78, 486 (1995)]. Other preferredmodifications include 2′-methoxy (2′—O—CH₃), 2′-propoxy (2′—OCH₂CH₂CH₃),2′-aminopropoxy (2′—OCH ₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow. Similar modifications may also be made at other positionson the oligonucleotide, particularly the 3′ position of the sugar on the3′ terminal nucleotide and the 5′ position of the 5′ terminalnucleotide. Oligonucleotides may also have sugar mimetics such ascyclobutyls in place of the pentofuranosyl group.

[0032] The oligonucleotides of the invention may additionally oralternatively include nucleobase modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include adenine (A),guanine (G), thymine (T), cytosine (C) and uracil (U). Modifiednucleobases include nucleobases found only infrequently or transientlyin natural nucleic acids, e.g., hypoxanthine, 6-methyladenine and5-methylcytosine, as well as synthetic nucleobases, e.g., 5-bromouracil,5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,N⁶(6-aminohexyl)adenine and 2,6-diaminopurine [Kornberg, A., DNAReplication, 1974, W. H. Freeman & Co., San Francisco, 1974, pp. 75-77;Gebeyehu, G., et al., Nucleic Acids Res., 15, 4513 (1987)].5-methylcytosine (5-me-C) is presently a preferred nucleobase,particularly in combination with 2′-O-methoxyethyl modifications.

[0033] Another preferred additional or alternative modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more lipophilic moieties which enhance thecellular uptake of the oligonucleotide. Such lipophilic moieties may belinked to an oligonucleotide at several different positions on theoligonucleotide. Some preferred positions include the 3′ position of thesugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the5′ terminal nucleotide, and the 2′ position of the sugar of anynucleotide. The N⁶ position of a purine nucleobase may also be utilizedto link a lipophilic moiety to an oligonucleotide of the invention(Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15, 4513). Suchlipophilic moieties include but are not limited to a cholesteryl moiety[Letsinger et al., Proc. Natl. Acad. Sci. USA,, 86, 6553 (1989)], cholicacid [Manoharan et al., Bioorg. Med. Chem. Let., 4, 1053 (1994)], athioether, e.g., hexyl-S-tritylthiol [Manoharan et al., Ann. N.Y. Acad.Sci., 660, 306 (1992); Manoharan et al., Bioorg. Med. Chem. Let., 3,2765 (1993)], a thiocholesterol [Oberhauser et al., Nucl. Acids Res.,20, 533 (1992)], an aliphatic chain, e.g., dodecandiol or undecylresidues [Saison-Behmoaras et al., EMBO J., 10, 111 (1991); Kabanov etal., FEES Lett., 259, 327 (1990); Svinarchuk et al., Biochimie., 75,49(1993)], a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate[Manoharan et al., Tetrahedron Lett., 36, 3651 (1995); Shea et al.,Nucl. Acids Res., 18, 3777 (1990)], a polyamine or a polyethylene glycolchain [Manoharan et al., Nucleosides & Nucleotides, 14, 969 (1995)], oradamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36, 3651(1995)], a palmityl moiety [Mishra et al., Biochim. Biophys. Acta, 1264,229 (1995)], or an octadecylamine or hexylamino-carbonyl-oxycholesterolmoiety [Crooke et al., J. Pharmacol. Exp. Ther., 277, 923 (1996)].Oligonucleotides comprising lipophilic moieties, and methods forpreparing such oligonucleotides, as disclosed in U.S. Pat. No.5,138,045, No. 5,218,105 and No. 5,459,255, the contents of which arehereby incorporated by reference.

[0034] The present invention also includes oligonucleotides which arechimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” inthe context of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art. ThisRNAse H-mediated cleavage of the RNA target is distinct from the use ofribozymes to cleave nucleic acids. Ribozymes are not comprehended by thepresent invention.

[0035] Examples of chimeric oligonucleotides include but are not limitedto “gapmers,” in which three distinct regions are present, normally witha central region flanked by two regions which are chemically equivalentto each other but distinct from the gap. A preferred example of a gapmeris an oligonucleotide in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for RNase H and is preferablycomposed of 2′-deoxynucleotides, while the flanking portions (the 5′ and3′ “wings”) are modified to have greater affinity for the target RNAmolecule but are unable to support nuclease activity (e.g., 2′-fluoro-or 2′-O-methoxyethyl-substituted). Other chimeras include “wingmers,”also known in the art as “hemimers,” that is, oligonucleotides with twodistinct regions. In a preferred example of a wingmer, the 5′ portion ofthe oligonucleotide serves as a substrate for RNase H and is preferablycomposed of 2′-deoxynucleotides, whereas the 3′ portion is modified insuch a fashion so as to have greater affinity for the target RNAmolecule but is unable to support nuclease activity (e.g., 2′-fluoro- or2′-O-methoxyethyl-substituted), or vice-versa. In one embodiment, theoligonucleotides of the present invention contain a 2′-O-methoxyethyl(2′—O—CH₂CH₂OCH₃) modification on the sugar moiety of at least onenucleotide. This modification has been shown to increase both affinityof the oligonucleotide for its target and nuclease resistance of theoligonucleotide. According to the invention, one, a plurality, or all ofthe nucleotide subunits of the oligonucleotides of the invention maybear a 2′-O-methoxyethyl (—O—CH₂CH₂OCH₃) modification. Oligonucleotidescomprising a plurality of nucleotide subunits having a 2′-O-methoxyethylmodification can have such a modification on any of the nucleotidesubunits within the oligonucleotide, and may be chimericoligonucleotides. Aside from or in addition to 2′-O-methoxyethylmodifications, oligonucleotides containing other modifications whichenhance antisense efficacy, potency or target affinity are alsopreferred. Chimeric oligonucleotides comprising one or more suchmodifications are presently preferred. Through use of suchmodifications, active oligonucleotides have been identified which areshorter than conventional “first generation” oligonucleotides activeagainst mdm2. Oligonucleotides in accordance with this invention arefrom 5 to 50 nucleotides in length, preferably from about 8 to about 30.In the context of this invention it is understood that this encompassesnon-naturally occurring oligomers as hereinbefore described, having from5 to 50 monomers, preferably from about 8 to about 30.

[0036] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives,including 2′-O-methoxyethyl oligonucleotides [Martin, P., Helv. Chim.Acta, 78, 486 (1995)]. It is also well known to use similar techniquesand commercially available modified amidites and controlled-pore glass(CPG) products such as biotin, fluorescein, acridine orpsoralen-modified amidites and/or CPG (available from Glen Research,Sterling, Va.) to synthesize fluorescently labeled, biotinylated orother conjugated oligonucleotides.

[0037] The antisense compounds of the present invention includebioequivalent compounds, including pharmaceutically acceptable salts andprodrugs. This is intended to encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of the nucleic acids of theinvention and prodrugs of such nucleic acids.

[0038] Pharmaceutically acceptable “salts” are physiologically andpharmaceutically acceptable salts of the nucleic acids of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto [see,for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci.,66:1 (1977)].

[0039] For oligonucleotides, examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; © salts formedwith organic acids such as, for example, acetic acid, oxalic acid,tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid,citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0040] The oligonucleotides of the invention may additionally oralternatively be prepared to be delivered in a “prodrug” form. The term“prodrug” indicates a therapeutic agent that is prepared in an inactiveform that is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

[0041] For therapeutic or prophylactic treatment, oligonucleotides areadministered in accordance with this invention. Oligonucleotidecompounds of the invention may be formulated in a pharmaceuticalcomposition, which may include pharmaceutically acceptable carriers,thickeners, diluents, buffers, preservatives, surface active agents,neutral or cationic lipids, lipid complexes, liposomes, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients and the like in addition to the oligonucleotide.Such compositions and formulations are comprehended by the presentinvention.

[0042] Pharmaceutical compositions comprising the oligonucleotides ofthe present invention may include penetration enhancers in order toenhance the alimentary delivery of the oligonucleotides. Penetrationenhancers may be classified as belonging to one of five broadcategories, i.e., fatty acids, bile salts, chelating agents, surfactantsand non-surfactants (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, 8:91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1). One or more penetrationenhancers from one or more of these broad categories may be included.Compositions comprising oligonucleotides and penetration enhancers aredisclosed in co-pending U.S. patent application Ser. No. 08/886,829 toTeng et al., filed Jul. 1, 1997, which is herein incorporated byreference in its entirety.

[0043] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional compatiblepharmaceutically-active materials such as, e.g., antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the composition of present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the invention.

[0044] Regardless of the method by which the oligonucleotides of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of theoligonucleotides and/or to target the oligonucleotides to a particularorgan, tissue or cell type. Colloidal dispersion systems include, butare not limited to, macromolecule complexes, nanocapsules, microspheres,beads and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, liposomes and lipid:oligonucleotide complexesof uncharacterized structure. A preferred colloidal dispersion system isa plurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layers made up of lipidsarranged in a bilayer configuration [see, generally, Chonn et al.,Current Op. Biotech., 6, 698 (1995)]. Liposomal antisense compositionsare prepared according to the disclosure of co-pending U.S. patentapplication Ser. No. 08/961,469 to Hardee et al., filed Oct. 31, 1997,herein incorporated by reference in its entirety.

[0045] The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous drip, subcutaneous, intraperitonealor intramuscular injection, pulmonary administration, e.g., byinhalation or insufflation, or intracranial, e.g., intrathecal orintraventricular, administration. Oligonucleotides with at least one2′-O-methoxyethyl modification are believed to be particularly usefulfor oral administration. Modes of administering oligonucleotides aredisclosed in co-pending U.S. patent application Ser. No. 08/961,469 toHardee et al., filed Oct. 31, 1997, herein incorporated by reference inits entirety.

[0046] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

[0047] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

[0048] Compositions for parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives. In some cases it may be more effective to treat apatient with an oligonucleotide of the invention in conjunction withother traditional therapeutic modalities in order to increase theefficacy of a treatment regimen. In the context of the invention, theterm “treatment regimen” is meant to encompass therapeutic, palliativeand prophylactic modalities. For example, a patient may be treated withconventional chemotherapeutic agents, particularly those used for tumorand cancer treatment. Examples of such chemotherapeutic agents includebut are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., eds.,Rahay, N.J., 1987). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

[0049] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0050] Thus, in the context of this invention, by “therapeuticallyeffective amount” is meant the amount of the compound which is requiredto have a therapeutic effect on the treated mammal. This amount, whichwill be apparent to the skilled artisan, will depend upon the type ofmammal, the age and weight of the mammal, the type of disease to betreated, perhaps even the gender of the mammal, and other factors whichare routinely taken into consideration when treating a mammal with adisease. A therapeutic effect is assessed in the mammal by measuring theeffect of the compound on the disease state in the animal. For example,if the disease to be treated is cancer, therapeutic effects are assessedby measuring the rate of growth or the size of the tumor, or bymeasuring the production of compounds such as cytokines, production ofwhich is an indication of the progress or regression of the tumor.

[0051] The following examples illustrate the present invention and arenot intended to limit the same.

EXAMPLES Example 1 Synthesis of Oligonucleotides

[0052] Unmodified oligodeoxynucleotides are synthesized on an automatedDNA synthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

[0053] 2′-methoxy oligonucleotides are synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base was increased to 360 seconds.Other 2′-alkoxy oligonucleotides were synthesized by a modification ofthis method, using appropriate 2′-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va.

[0054] 2′-fluoro oligonucleotides were synthesized as described inKawasaki et al., J. Med. Chem., 36, 831 (1993). Briefly, the protectednucleoside N⁶-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-β-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-α-fluoro atom is introduced by a S_(N)2-displacement of a2′-β-O-trifyl group. Thus N⁶-benzoyl-9-β-D-arabinofuranosyladenine wasselectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl(THP) intermediate. Deprotection of the THP and N⁶-benzoyl groups wasaccomplished using standard methodologies and standard methods were usedto obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramiditeintermediates.

[0055] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-β-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0056] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a known procedure in which 2,2′-anhydro-1-β-D-arabinofuranosyluracil was treated with 70% hydrogenfluoride-pyridine. Standard procedures were used to obtain the 5′-DMTand 5′-DMT-3′phosphoramidites.

[0057] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN⁴-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0058] 2′-(2-methoxyethyl)-modified amidites are synthesized accordingto Martin, P., Helv. Chim. Acta, 78,486 (1995). For ease of synthesis,the last nucleotide was a deoxynucleotide. 2′—O—CH₂CH₂OCH₃-cytosines maybe 5-methyl cytosines.

Synthesis of 5-Methyl Cytosine Monomers

[0059] 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:

[0060] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 hours) to give a solid which was crushed to a light tanpowder (57 g, 85% crude yield). The material was used as is for furtherreactions.

2′-O-Methoxyethyl-5-methyluridine

[0061] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product.

2-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0062] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxy-trityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/-Hexane/Acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-uridine

[0063] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tlc by first quenching the tlc sample with the additionof MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0064] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0065] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine

[0066] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tlc showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0067]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL) , and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0068] 5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotideswere synthesized according to published methods [Sanghvi et al., Nucl.Acids Res., 21, 3197 (1993)] using commercially availablephosphoramidites (Glen Research, Sterling, Va. or ChemGenes, Needham,Mass.).

2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0069] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0070] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0071] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig) . The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0072]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0073]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 hr the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eg.) was addedand the mixture for 1 hr. Solvent was removed under vacuum; residuechromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0074]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 hr, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL) . Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0075] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂) Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0076] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0077] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) Nucleoside Amidites

[0078] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0079] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced byN-hydroxyphthalimide via a Mitsunobu reaction, and the protectednucleoside may phosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0080] Oligonucleotides having methylene (methylimino) (MMI) backbonesare synthesized according to U.S. Pat. No. 5,378,825, which iscoassigned to the assignee of the present invention and is incorporatedherein in its entirety. For ease of synthesis, various nucleoside dimerscontaining MMI linkages were synthesized and incorporated intooligonucleotides. Other nitrogen-containing backbones are synthesizedaccording to WO 92/20823 which is also coassigned to the assignee of thepresent invention and incorporated herein in its entirety.

[0081] Oligonucleotides having amide backbones are synthesized accordingto De Mesmaeker et al., Acc. Chem. Res., 28, 366 (1995). The amidemoiety is readily accessible by simple and well-known synthetic methodsand is compatible with the conditions required for solid phase synthesisof oligonucleotides.

[0082] Oligonucleotides with morpholino backbones are synthesizedaccording to U.S. Pat. No. 5,034,506 (Summerton and Weller).

[0083] Peptide-nucleic acid (PNA) oligomers are synthesized according toP. E. Nielsen et al., Science, 254, 1497 (1991).

[0084] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsand judged to be at least 85% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in synthesiswere periodically checked by ³¹p nuclear magnetic resonancespectroscopy, and for some studies oligonucleotides were purified byHPLC, as described by Chiang et al., J. Biol. Chem., 266, 18162 (1991).Results obtained with HPLC-purified material were similar to thoseobtained with non-HPLC purified material.

Example 2 Human mdm2 Oligonucleotide Sequences

[0085] The oligonucleotides tested are presented in Table 1. Sequencedata are from the cDNA sequence published by Oliner, J. D., et al.,Nature, 358, 80 (1992); Genbank accession number Z12020, provided hereinas SEQ ID NO: 1. Oligonucleotides were synthesized primarily as chimericoligonucleotides having a centered deoxy gap of eight nucleotidesflanked by 2′-O-methoxyethyl regions.

[0086] A549 human lung carcinoma cells (American Type CultureCollection, Manassas, Va.) were routinely passaged at 80-90% confluencyin Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum(Hyclone, Logan, Utah). JEG-3 cells, a human choriocarcinoma cell line(American Type Culture Collection, Manassas, Va.), were maintained inRPMI1640, supplemented with 10% fetal calf serum. All cell culturereagents, except as otherwise indicated, are obtained from LifeTechnologies (Rockville, Md.).

[0087] A549 cells were treated with phosphorothioate oligonucleotides at200 nM for four hours in the presence of 6 μg/ml LIPOFECTIN™, washed andallowed to recover for an additional 20 hours. Total RNA was extractedand 15-20 μg of each was resolved on 1% gels and transferred to nylonmembranes. The blots were probed with a ³²p radiolabeled mdm2 cDNA probeand then stripped and reprobed with a radiolabeled G3PDH probe toconfirm equal RNA loading. mdm2 transcripts were examined and quantifiedwith a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). Resultsare shown in Table 2. Oligonucleotides 16506 (SEQ ID NO: 3), 16507 (SEQID NO: 4), 16508 (SEQ ID NO: 5), 16510 (SEQ ID NO: 7), 16518 (SEQ ID NO:15), 16520 (SEQ ID NO: 17), 16521 (SEQ ID NO: 18), 16522 (SEQ ID NO: 19)and 16524 (SEQ ID NO: 21) gave at least approximately 50% reduction ofmdm2 mRNA levels. Oligonucleotides 16507 and 16518 gave better than 85%reduction of mdm2. TABLE 1 Nucleotide Sequences of Human mdm2Phosphorothioate Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDESEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION16506 CAGCCAAGCTCGCGCGGTGC 3 0001-0020 5′-UTR 16507 TCTTTCCGACACACAGGGCC4 0037-0056 5′-UTR 16508 CAGCAGGATCTCGGTCAGAG 5 0095-0114 5′-UTR 16509GGGCGCTCGTACGCACTAAT 6 0147-0166 5′-UTR 16510 TCGGGGATCATTCCACTCTC 70181-0200 5′-UTR 16511 CGGGGTTTTCGCGCTTGGAG 8 0273-0292 5′-UTR 16512CATTTGCCTGCTCCTCACCA 9 0295-0314 AUG 16513 GTATTGCACATTTGCCTGCT 100303-0322 AUG 16514 AGCACCATCAGTAGGTACAG 11 0331-0350 ORF 16515CTACCAAGTTCCTGTAGATC 12 0617-0636 ORF 16516 TCAACTTCAAATTCTACACT 131047-1066 ORF 16517 TTTACAATCAGGAACATCAA 14 1381-1400 ORF 16518AGCTTCTTTGCACATGTAAA 15 1695-1714 ORF 16519 CAGGTCAACTAGGGGAAATA 161776-1795 stop 16520 TCTTATAGACAGGTCAACTA 17 1785-1804 stop 16521TCCTAGGGTTATATAGTTAG 18 1818-1837 3′-UTR 16522 AAGTATTCACTATTCCACTA 191934-1953 3′-UTR 16523 CCAAGATCACCCACTGCACT 20 2132-2151 3′-UTR 16524AGGTGTGGTGGCAGATGACT 21 2224-2243 3′-UTR 16525 CCTGTCTCTACTAAAAGTAC 222256-2275 3′-UTR 17604 ACAAGCCTTCGCTCTACCGG 23 scrambled 16507 control17605 TTCAGCGCATTTGTACATAA 24 scrambled 16518 control 17615TCTTTCCGACACACAGGGCC 25 0037-0056 5′-UTR 17616 AGCTTCTTTGCACATGTAAA 151695-1714 ORF 17755 AGCTTCTTTGCACATGTAAA 15 1695-1714 ORF 17756AGCTTCTTTATACATGTAAA 26 2-base 17616 mismatch 17757 AGCTTCTTTACACATGTAAA27 1-base 17616 mismatch # 267-286 on the Landers sequence.

[0088] TABLE 2 Activities of Phosphorothioate Oligonucleotides Targetedto Human mdm2 SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO: REGIONEXPRESSION INHIBITION LIPOFECTIN ™ — — 100% 0% only 16506 3 5′-UTR 45%55% 16507 4 5′-UTR 13% 87% 16508 5 5′-UTR 38% 62% 16509 6 5′-UTR 161% —16510 7 5′-UTR 46% 54% 16511 8 5′-UTR 91% 9% 16512 9 AUG 89% 11% 1651310 AUG 174% — 16514 11 Coding 92% 8% 16515 12 Coding 155% — 16516 13Coding 144% — 16517 14 Coding 94% 6% 16518 15 Coding 8% 92% 16519 16stop 73% 27% 16520 17 stop 51% 49% 16521 18 3′-UTR 38% 62% 16522 193′-UTR 49% 51% 16523 20 3′-UTR 109% — 16524 21 3′-UTR 47% 53% 16525 223′-UTR 100% —

Example 3 Dose Response of Antisense Oligonucleotide Effects on Humanmdm2 mRNA Levels in A549 Cells

[0089] Oligonucleotides 16507 and 16518 were tested at differentconcentrations. A549 cells were grown, treated and processed asdescribed in Example 2. LIPOFECTIN™ was added at a ratio of 3 μg/ml per100 nM of oligonucleotide. The control included LIPOFECTINT™ at aconcentration of 12 μg/ml. Oligonucleotide 17605, an oligonucleotidewith different sequence but identical base composition tooligonucleotide 16518, was used as a negative control. Results are shownin Table 3. Oligonucleotides 16507 and 16518 gave approximately 90%inhibition at concentrations greater than 200 nM. No inhibition was seenwith oligonucleotide 17605. TABLE 3 Dose Response of A549 Cells to mdm2Antisense Oligonucleotides (ASOs) SEQ % mRNA % mRNA ID ASO Gene Ex- In-ISIS # NO: Target Dose pression hibition control — LIPOFECTIN ™ — 100%0% only 16507 4 5′-UTR  25 nM 55% 45% 16507 4 ″  50 nM 52% 48% 16507 4 ″100 nM 24% 76% 16507 4 ″ 200 nM 12% 88% 16518 15 Coding  50 nM 18% 82%16518 15 ″ 100 nM 14% 86% 16518 15 ″ 200 nM 9% 91% 16518 15 ″ 400 nM 8%92% 17605 24 scrambled 400 nM 129% —

Example 4 Time Course of Antisense Oligonucleotide Effects on Human mdm2mRNA Levels in A549 Cells

[0090] Oligonucleotides 16507 and 17605 were tested by treating forvarying times. A549 cells were grown, treated for times indicated inTable 4 and processed as described in Example 2. Results are shown inTable 4. Oligonucleotide 16507 gave greater than 90% inhibitionthroughout the time course. No inhibition was seen with oligonucleotide17605. TABLE 4 Time Course of Response of Cells to Human mdm2 AntisenseOligonucleotides (ASOs) SEQ ASO Gene % RNA % RNA ID Target Ex- In- ISIS# NO: Region Time pression hibition basal — LIPOFECTIN ™ 24 h 100% 0%only basal — LIPOFECTIN ™ 48 h 100% 0% only basal — LIPOFECTIN ™ 72 h100% 0% only 16518 15 Coding 24 h 3% 97% 16518 15 ″ 48 h 6% 94% 16518 15″ 72 h 5% 95% 17605 24 scrambled 24 h 195% — 17605 24 ″ 48 h 100% —17605 24 ″ 72 h 102% —

Example 5 Effect of Antisense Oligonucleotides on Cell Proliferation inA549 Cells

[0091] A549 cells were treated on day 0 for four hours with 400 nMoligonucleotide and 12 mg/ml LIPOFECTIN. After four hours, the mediumwas replaced. Twenty-four, forty-eight or seventy-two hours afterinitiation of oligonucleotide treatment, live cells were counted on ahemacytometer. Results are shown in Table 5. TABLE 5 AntisenseInhibition of Cell Proliferation in A549 cells SEQ ASO Gene ID Target %Cell % Growth ISIS # NO: Region Time Growth Inhibition basal —LIPOFECTIN ™ 24 h 100% 0% only basal — LIPOFECTIN ™ 48 h 100% 0% onlybasal — LIPOFECTIN ™ 72 h 100% 0% only 16518 15 Coding 24 h 53% 47%16518 15 ″ 48 h 27% 73% 16518 15 ″ 72 h 17% 83% 17605 24 scrambled 24 h93% 7% 17605 24 ″ 48 h 76% 24% 17605 24 ″ 72 h 95% 5%

Example 6 Effect of mdm2 Antisense Oligonucleotide on p53 Protein Levels

[0092] JEG3 cells were cultured and treated as described in Example 2,except that 300 nM oligonucleotide and 9 μg/ml of LIPOFECTIN™ was used.

[0093] For determination of p53 protein levels by western blot, cellularextracts were prepared using 300 μl of RIPA extraction buffer per 100-mmdish. The protein concentration was quantified by Bradford assay usingthe BioRad kit (BioRad, Hercules, Calif.). Equal amounts of protein wereloaded on 10% or 12% SDS-PAGE mini-gel (Novex, San Diego, Calif.). Oncetransferred to PVDF membranes (Millipore, Bedford, Mass.), the membraneswere then treated for a minimum of 2h with specific primary antibody(p53 antibody, Transduction Laboratories, Lexington, Ky.) followed byincubation with secondary antibody conjugated to HRP. The results werevisualized by ECL Plus Western Blotting Detection System (AmershamPharmacia Biotech, Piscataway, N.J.). In some experiments, the blotswere stripped in stripping buffer (2% SDS, 12.5 mM Tris, pH 6.8) for 30min. at 50° C. After extensive washing, the blots were blocked andblotted with different primary antibody.

[0094] Results are shown in Table 6. Treatment with mdm2 antisenseoligonucleotide results in the induction of p53 levels. An approximatelythree-fold increase in activity was seen under these conditions. TABLE 6Activity of ISIS 16518 on p53 Protein Levels GENE ISIS SEQ ID TARGET %protein No: NO: REGION EXPRESSION LIPOFECTIN ™ only — — 100% 16518 15coding 289%

Example 7 Effect of ISIS 16518 on Expression of p53 Mediated Genes

[0095] p53 is known to regulate the expression of a number of genes andto be involved in apoptosis. Representative genes known to be regulatedby p53 include p21 (Deng, C., et al., Cell, 1995, 82, 675), bax(Selvakumaran, M., et al., Oncogene, 1994, 9, 1791-1798) and GADD45(Carrier, F., et al., J. Biol. Chem., 1994, 269, 32672-32677). Theeffect of an mdm2 antisense oligonucleotide on these genes isinvestigated by RPA analysis using the RIBOQUANT™ RPA kit, according tothe manufacturer's instructions (Pharmingen, San Diego, Calif.), alongwith the hSTRESS-1 multi-probe template set. Included in this templateset are bclx, p53, GADD45, c-fos, p21, bax, bcl2 and mcl1. The effect ofmdm2 antisense oligonucleotides on p53-mediated apoptosis can readily beassessed using commercial kits based on apoptotic markers such as DNAfragmentation or caspase activity.

Example 8 Additional Human mdm2 Chimeric (Deoxy Gapped) AntisenseOligonucleotides

[0096] Additional oligonucleotides targeted to the 5′-untranslatedregion of human mdm2 mRNA were designed and synthesized. Sequence dataare from the cDNA sequence published by Zauberman, A., et al., NucleicAcids Res., 23, 2584 (1995); Genbank accession number HSU28935.Oligonucleotides were synthesized primarily as chimeric oligonucleotideshaving a centered deoxy gap of eight nucleotides flanked by2′-O-methoxyethyl regions. The oligonucleotide sequences are shown inTable 7. These oligonucleotides were tested in A549 cells as describedin Example 2. Results are shown in Table 8. TABLE 7 Nucleotide Sequencesof additional Human mdm2 Chimeric (deoxy gapped) PhosphorothioateOligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ IDNUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 21926CTACCCTCCAATCGCCACTG 28 0238-0257 coding 21927 GGTCTACCCTCCAATCGCCA 290241-0260 coding 21928 CGTGCCCACAGGTCTACCCT 30 0251-0270 coding 21929AAGTGGCGTGCGTCCGTGCC 31 0265-0284 coding 21930 AAAGTGGCGTGCGTCCGTGC 320266-0285 coding

[0097] TABLE 8 Activities of Chimeric (deoxy gapped) OligonucleotidesTargeted to Human mdm2 SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO:REGION EXPRESSION INHIBITION LIPOFECTIN ™ — — 100% 0% only 21926 28coding 345% — 21927 29 coding 500% — 21928 30 coding 417% — 21929 31coding 61% 39% 21930 32 coding 69% 31%

[0098] These oligonucleotide sequences were also tested for theirability to reduce mdm2 protein levels. JEG3 cells were cultured andtreated as described in Example 2, except that 300 nM oligonucleotideand 9 μg/ml of LIPOFECTIN™ was used. Mdm2 protein levels were assayed byWestern blotting as described in Example 6, except an mouse anti-mdm2monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) wasused. Results are shown in Table 9. TABLE 9 Activities of Chimeric(deoxy gapped) Human mdm2 Antisense Oligonucleotides on mdm2 ProteinLevels SEQ GENE ISIS ID TARGET % PROTEIN % PROTEIN No: NO: REGIONEXPRESSION INHIBITION LIPOFECTIN ™ — — 100% 0% only 21926 28 coding 30%70% 21927 29 coding 18% 82% 21928 30 coding 43% 57% 21929 31 coding 62%38% 21930 32 coding 56% 44%

[0099] Each oligonucleotide tested reduced mdm2 protein levels bygreater than approximately 40%. Maximum inhibition was seen witholigonucleotide 21927 (SEQ ID NO. 29) which gave greater than 80%inhibition of mdm2 protein.

Example 9 Additional Human mdm2 Antisense Oligonucleotides

[0100] Additional oligonucleotides targeted to human mdm2 mRNA weredesigned and synthesized. Sequence data are from the cDNA sequencepublished by Zauberman, A., et al., Nucleic Acids Res., 23, 2584 (1995);Genbank accession number HSU28935. Oligonucleotides were synthesized in96 well plate format via solid phase P(III) phosphoramidite chemistry onan automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-di-isopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per published methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0101] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

[0102] Two sets of oligonucleotides were synthesized; one asphosphorothioate oligodeoxynucleotides, the other as chimericoligonucleotides having a centered deoxy gap of ten nucleotides flankedby regions of five 2′-O-methoxyethyl nucleotides. These oligonucleotidessequences are shown in Tables 10 and 11.

[0103] mRNA was isolated using the RNAEASY® kit (Qiagen, Santa Clarita,Calif.). TABLE 10 Nucleotide Sequences of Human mdm2 PhosphorothioateOligodeoxynucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ IDNUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES² REGION 31393CAGCCAAGCTCGCGCGGTGC 3 0001-0020 5′ UTR 31712 AAGCAGCCAAGCTCGCGCGG 330004-0023 5′ UTR 31552 CAGGCCCCAGAAGCAGCCAA 34 0014-0033 5′ UTR 31713GCCACACAGGCCCCAGAAGC 35 0020-0039 5′ UTR 31394 ACACACAGGGCCACACAGGC 360029-0048 5′ UTR 31714 TTCCGACACACAGGGCCACA 37 0034-0053 5′ UTR 31553GCTCCATCTTTCCGACACAC 38 0043-0062 5′ UTR 31715 GCTTCTTGCTCCATCTTTCC 390050-0069 5′ UTR 31395 CCCTCGGGCTCGGCTTCTTG 40 0062-0081 5′ UTR 31716GCGGCCGCCCCTCGGGCTCG 41 0070-0089 5′ UTR 31554 AAGCAGCAGGATCTCGGTCA 420098-0107 5′ UTR 31717 GCTGCGAAAGCAGCAGGATC 43 0105-0124 5′ UTR 31396TGCTCCTGGCTGCGAAAGCA 44 0113-0132 5′ UTR 31718 GGGACGGTGCTCCTGGCTGC 450120-0139 5′ UTR 31555 ACTGGGCGCTCGTACGCACT 46 0150-0169 5′ UTR 31719GCCAGGGCACTGGGCGCTCG 47 0158-0177 5′-UTR 31397 TCTCCGGGCCAGGGCACTGG 480165-0184 5′ UTR 31720 TCATTCCACTCTCCGGGCCA 49 0174-0193 5′ UTR 31556GGAAGCACGACGCCCTGGGC 50 0202-0221 5′ UTR 31721 TACTGCGGAAGCACGACGCC 510208-0227 5′ UTR 31398 GGGACTGACTACTGCGGAAG 52 0217-0236 5′ UTR 31722TCAAGACTCCCCAGTTTCCT 53 0242-0261 5′ UTR 31557 CCTGCTCCTCACCATCCGGG 540289-0308 5′ UTR 31399 TTTGCCTGCTCCTCACCATC 55 0293-0312 AUG 31400ATTTGCCTGCTCCTCACCAT 56 0294-0313 AUG 31401 CATTTGCCTGCTCCTCACCA 90295-0314 AUG 31402 ACATTTGCCTGCTCCTCACC 57 0296-0315 AUG 31403CACATTTGCCTGCTCCTCAC 58 0297-0316 AUG 31404 GCACATTTGCCTGCTCCTCA 590298-0317 AUG 31405 TGCACATTTGCCTGCTCCTC 60 0299-0318 AUG 31406TTGCACATTTGCCTGCTCCT 61 0300-0319 AUG 31407 ATTGCACATTTGCCTGCTCC 620301-0320 AUG 31408 TATTGCACATTTGCCTGCTC 63 0302-0321 AUG 31409GTATTGCACATTTGCCTGCT 10 0303-0322 AUG 31410 GGTATTGCACATTTGCCTGC 640304-0323 AUG 31411 TGGTATTGCACATTTGCCTG 65 0305-0324 AUG 31412TTGGTATTGCACATTTGCCT 66 0306-0325 AUG 31413 GTTGGTATTGCACATTTGCC 670307-0326 AUG 31414 TGTTGGTATTGCACATTTGC 68 0308-0327 AUG 31415ATGTTGGTATTGCACATTTG 69 0309-0328 AUG 31416 CATGTTGGTATTGCACATTT 700310-0329 AUG 31417 ACATGTTGGTATTGCACATT 71 0311-0330 AUG 31418GACATGTTGGTATTGCACAT 72 0312-0331 AUG 31419 AGACATGTTGGTATTGCACA 730313-0332 AUG 31420 CAGACATGTTGGTATTGCAC 74 0314-0333 AUG 31558CAGTAGGTACAGACATGTTG 75 0323-0342 coding 31723 TACAGCACCATCAGTAGGTA 760334-0353 coding 31421 GGAATCTGTGAGGTGGTTAC 77 0351-0370 coding 31559TTCCGAAGCTGGAATCTGTG 78 0361-0380 coding 31724 AGGGTCTCTTGTTCCGAAGC 790372-0391 coding 31422 GCTTTGGTCTAACCAGGGTC 80 0386-0405 coding 31560GCAATGGCTTTGGTCTAACC 81 0392-0411 coding 31725 TAACTTCAAAAGCAATGGCT 820403-0422 coding 31423 GTGCACCAACAGACTTTAAT 83 0422-0441 coding 31561ACCTCTTTCATAGTATAAGT 84 0450-0469 coding 31726 ATAATATACTGGCCAAGATA 850477-0496 coding 31424 TAATCGTTTAGTCATAATAT 86 0490-0509 coding 31727ATCATATAATCGTTTAGTCA 87 0496-0515 coding 31562 GCTTCTCATCATATAATCGT 880503-0522 coding 31728 CAATATGTTGTTGCTTCTCA 89 0515-0534 coding 31425GAACAATATACAATATGTTG 90 0525-0544 coding 31729 TCATTTGAACAATATACAAT 910531-0550 coding 31563 TAGAAGATCATTTGAACAAT 92 0538-0557 coding 31730AACAAATCTCCTAGAAGATC 93 0549-0568 coding 31426 TGGCACGCCAAACAAATCTC 940559-0578 coding 31731 AGAAGCTTGGCACGCCAAAC 95 0566-0585 coding 31564CTTTCACAGAGAAGCTTGGC 96 0575-0594 coding 31732 TTTTCCTGTGCTCTTTCACA 970587-0606 coding 31427 TATATATTTTCCTGTGCTCT 98 0593-0612 coding 31733ATCATGGTATATATTTTCCT 99 0600-0619 coding 31565 TTCCTGTAGATCATGGTATA 1000609-0628 coding 31734 TACTACCAAGTTCCTGTAGA 101 0619-0638 coding 31428TTCCTGCTGATTGACTACTA 102 0634-0653 coding 31566 TGAGTCCGATGATTCCTGCT 1030646-0665 coding 31735 CAGATGTACCTGAGTCCGAT 104 0656-0675 coding 31429CTGTTCTCACTCACAGATGT 105 0669-0688 coding 31567 TTCAAGGTGACACCTGTTCT 1060682-0701 coding 31736 ACTCCCACCTTCAAGGTGAC 107 0691-0710 coding 31430GGTCCTTTTGATCACTCCCA 108 0704-0723 coding 31568 AAGCTCTTGTACAAGGTCCT 1090718-0737 coding 31737 CTCTTCCTGAAGCTCTTGTA 110 0727-0746 coding 31431AAGATGAAGGTTTCTCTTCC 111 0740-0759 coding 31569 AAACCAAATGTGAAGATCAA 1120752-0771 coding 31738 ATGGTCTAGAAACCAAATGT 113 0761-0780 coding 31432CTAGATGAGGTAGATGGTCT 114 0774-0793 coding 31570 AATTGCTCTCCTTCTAGATG 1150787-0806 coding 31739 TCTGTCTCACTAATTGCTCT 116 0798-0817 coding 31433TCTGAATTTTCTTCTGTCTC 117 0810-0829 coding 31571 CACCAGATAATTCATCTCAA 1180824-0843 coding 31740 TTTGTCGTTCACCAGATAAT 119 0833-0852 coding 31434GTGGCGTTTTCTTTGTCGTT 120 0844-0863 coding 31572 TACTATCAGATTTGTGGCGT 1210857-0876 coding 31741 GAAAGGCAAATACTATCACA 122 0867-0886 coding 31435GCTTTCATCAAAGGAAAGGG 123 0880-0899 coding 31573 TACACACAGAGCCAGGCTTT 1240895-0914 coding 31742 CTCCCTTATTACACACAGAG 125 0904-0923 coding 31436TCACAACATATCTCCCTTAT 126 0915-0934 coding 31574 CTACTGCTTCTTTCACAACA 1270927-0946 coding 31743 GATTCACTGCTACTGCTTCT 128 0936-0955 coding 31437TGGCGTCCCTGTAGATTCAC 129 0949-0968 coding 31575 AAGATCCGGATTCGATGGCG 1300964-0983 coding 31744 CAGCATCAAGATCCGGATTC 131 0971-0990 coding 31438GTTCACTTACACCAGCATCA 132 0983-1002 coding 31576 CAATCACCTGAATGTTCACT 1330996-1015 coding 31745 CTGATCCAACCAATCACCTG 134 1006-1025 coding 31439GAAACTGAATCCTGATCCAA 135 1017-1036 coding 31746 TGATCTGAAACTGAATCCTG 1361023-1042 coding 31577 CTACACTAAACTGATCTGAA 137 1034-1053 coding 31747CAACTTCAAATTCTACACTA 138 1046-1065 coding 31440 AGATTCAACTTCAAATTCTA 1391051-1070 coding 31748 GAGTCGAGAGATTCAACTTC 140 1059-1078 coding 31578TAATCTTCTGAGTCGAGAGA 141 1068-1087 coding 31749 CTAAGGCTATAATCTTCTGA 1421077-1096 coding 31441 TTCTTCACTAAGGCTATAAT 143 1084-1103 coding 31750TCTTGTCCTTCTTCACTAAG 144 1092-1111 coding 31579 CTGAGAGTTCTTGTCCTTCT 1451100-1119 coding 31751 TTCATCTGAGAGTTCTTGTC 146 1105-1124 coding 31442CCTCATCATCTTCATCTGAG 147 1115-1134 coding 31752 CTTGATATACCTCATCATCT 1481124-1143 coding 31753 ATACACAGTAA(TTGATATA 149 1135-1154 coding 31443CTCTCCCCTGCCTGATACAC 150 1149-1168 coding 31580 GAATCTGTATCACTCTCCCC 1511161-1180 coding 31754 TCTTCAAATGAATCTGTATC 152 1170-1189 coding 31444AAATTTCAGGATCTTCTTCA 153 1184-1203 coding 31581 AGTCAGCTAAGGAAATTTCA 1541196-1215 coding 31755 GCATTTCCAATAGTCAGCTA 155 1207-1226 coding 31445CATTGCATGAAGTGCATTTC 156 1220-1239 coding 31756 TCATTTCATTGCATGAAGTG 1571226-1245 coding 31582 CATCTGTTGCAATGTGATGG 158 1257-1276 coding 31757GAAGGGCCCAACATCTGTTG 159 1268-1287 coding 31446 TTCTCACGAAGGGCCCAACA 1601275-1294 coding 31758 GAAGCCAATTCTCACGAAGG 161 1283-1302 coding 31583TATCTTCAGGAAGCCAATTC 162 1292-1311 coding 31759 CTTTCCCTTTATCTTCAGGA 1631301-1320 coding 31447 TCCCCTTTATCTTTCCCTTT 164 1311-1330 coding 31584CTTTCTCAGAGATTTCCCCT 165 1325-1344 coding 31760 CAGTTTGGCTTTCTCAGAGA 1661333-1352 coding 31448 GTGTTGAGTTTTCCAGTTTG 167 1346-1365 coding 31585CCTCTTCAGCTTGTGTTGAG 168 1358-1377 coding 31761 ACATCAAAGCCCTCTTCAGC 1691368-1787 coding 31449 GAATCATTCACTATAGTTTT 170 1401-1420 coding 31586ATGACTCTCTGGAATCATTC 171 1412-1431 coding 31762 CCTCAACACATGACTCTCTG 1721421-1440 coding 31450 TTATCATCATTTTCCTCAAC 173 1434-1453 coding 31763TAATTTTATCATCATTTTCC 174 1439-1458 coding 31587 GAAGCTTGTGTAATTTTATC 1751449-1468 coding 31764 TGATTGTGAAGCTTGTGTAA 176 1456-1475 coding 31451CACTTTCTTGTGATTGTGAA 177 1466-1485 coding 31588 GCTGAGAATAGTCTTCACTT 1781481-1500 coding 31765 AGTTGATGGCTGAGAATAGT 179 1489-1508 coding 31452TGCTACTAGAAGTTGATGGC 180 1499-1518 coding 31766 TAAATAATGCTACTAGAAGT 1811506-1525 coding 31589 CTTGGCTGCTATAAATAATG 182 1517-1536 coding 31590ATCTTCTTGGCTGCTATAAA 183 1522-1541 coding 31453 AACTCTTTCACATCTTCTTG 1841533-1552 coding 31767 CCCTTTCAAACTCTTTCACA 185 1541-1560 coding 31591GGGTTTCTTCCCTTTCAAAC 186 1550-1569 coding 31768 TCTTTGTCTTGGGTTTCTTC 1871560-1579 coding 31454 CTCTCTTCTTTGTCTTGGGT 188 1566-1585 coding 31592AACTAGATTCCACACTCTCT 189 1580-1599 coding 31769 CAAGGTTCAATGGCATTAAG 1901605-1624 coding 31455 TGACAAATCACACAAGGTTC 191 1617-1636 coding 31593TCGACCTTGACAAATCACAC 192 1624-1643 coding 31594 ATGGACAATGCAACCATTTT 1931648-1667 coding 31770 TGTTTTGCCATGGACAATGC 194 1657-1676 coding 31456TAAGATGTCCTGTTTTGCCA 195 1667-1686 coding 31595 GCAGGCCATAAGATGTCCTG 1961675-1694 coding 31596 ACATGTAAAGCAGGCCATAA 197 1684-1703 coding 31771CTTTGCACATGTAAAGCAGG 198 1690-1709 coding 31457 TTTCTTTAGCTTCTTTGCAC 1991702-1721 coding 31597 TTATTCCTTTTCTTTAGCTT 200 1710-1729 coding 31598TGGGCAGGGCTTATTCCTTT 201 1720-1739 coding 31772 ACATACTGGGCAGGGCTTAT 2021726-1745 coding 31458 TTGGTTGTCTACATACTGGG 203 1736-1755 coding 31599TCATTTGAATTGGTTGTCTA 204 1745-1764 coding 31600 AAGTTAGCACAATCATTTGA 2051757-1776 coding 31601 TCTCTTATAGACAGGTCAAC 206 1787-1806 STOP 31459AAATATATAATTCTCTTATA 207 1798-1817 3′ UTR 31602 AGTTAGAAATATATAATTCT 2081804-1823 3′ UTR 31773 ATATAGTTAGAAATATATAA 209 1808-1827 3′ UTR 31603CTAGGGTTATATAGTTAGAA 210 1816-1835 3′ UTR 31774 TAAATTCCTAGGGTTATATA 2111823-1842 3′ UTR 31460 CAGGTTGTCTAAATTCCTAG 212 1832-1851 3′ UTR 31604ATAAATTTCAGGTTGTCTAA 213 1840-1859 3′ UTR 31605 ATATATGTGAATAAATTTCA 2141850-1869 3′ UTR 31606 CTTTGATATATGTGAATAAA 215 1855-1874 3′ UTR 31461CATTTTCTCACTTTGATATA 216 1865-1884 3′ UTR 31607 ATTGAGGCATTTTCTCACTT 2171872-1891 3′ UTR 31608 AATCTATGTGAATTGAGGCA 218 1883-1902 3′ UTR 31609AGAAGAAATCTATGTGAATT 219 1889-1908 3′ UTR 31462 ATACTAAAGAGAAGAAATCT 2201898-1917 3′ UTR 31610 GTCAATTATACTAAAGAGAA 221 1905-1924 3′ UTR 31775TAGGTCAATTATACTAAAGA 222 1908-1927 3′ UTR 31611 CAAAGTAGGTCAATTATACT 2231913-1932 3′ UTR 31776 CCACTACCAAAGTAGGTCAA 224 1920-1939 3′ UTR 31463AGTATTCACTATTCCACTAC 225 1933-1952 3′ UTR 31612 TATAGTAAGTATTCACTATT 2261940-1959 3′ UTR 31613 AGTCAAATTATAGTAAGTAT 227 1948-1967 3′ UTR 31777CATATTCAAGTCAAATTATA 228 1956-1975 3′ UTR 31464 AAAGGATGAGCTACATATTC 2291969-1988 3′ UTR 31778 GTGTAAAGGATGAGCTACAT 230 1973-1992 3′ UTR 31614TAGGAGTTGGTGTAAAGGAT 231 1982-2001 3′ UTR 31779 TTTAAAATTAGGAGTTGGTG 2321990-2009 3′ UTR 31615 GAAATTATTTAAAATTAGGA 233 1997-2016 3′ UTR 31465CAGAGTAGAAATTATTTAAA 234 2004-2023 3′ UTR 31616 CTCATTTAAGACAGAGTAGA 2352015-2034 3′ UTR 31780 TACTTCTCATTTAAGACAGA 236 2020-2039 3′ UTR 31617CATATACATATTTAAGAAAA 237 2051-2070 3′ UTR 31466 TTAAATGTCATATACATATT 2382059-2078 3′ UTR 31618 TAATAAGTTACATTTAAATG 239 2072-2091 3′ UTR 31619GTAACAGAGCAAGACTCGGT 240 2103-2122 3′ UTR 31467 CAGCCTGGGTAACAGAGCAA 2412111-2130 3′ UTR 31781 CACTCCAGCCTGGGTAACAG 242 2116-2135 3′ UTR 31620CCCACTGCACTCCAGCCTGG 243 2123-2142 3′ UTR 31782 GCCAAGATCACCCACTGCAC 2442133-2152 3′ UTR 31621 GCAGTGAGCCAAGATCACCC 245 2140-2159 3′ UTR 31468GAGCTTGCAGTGAGCCAAGA 246 2146-2165 3′ UTR 31783 GAGGGCAGAGCTTGCAGTGA 2472153-2172 3′ UTR 31622 CAGGAGAATGGTGCGAACCC 248 2176-2195 3′ UTR 31623AGGCTGAGGCAGGAGAATGG 249 2185-2204 3′ UTR 31784 ATTGGGAGGCTGAGGCAGGA 2502191-2210 3′ UTR 31469 CAAGCTAATTGGGAGGCTGA 251 2198-2217 3′ UTR 31624AGGCCAAGCTAATTGGGAGG 252 2202-2221 3′ UTR 31785 ATGACTGTAGGCCAAGCTAA 2532210-2229 3′ UTR 31625 CAGATGACTGTAGGCCAAGC 254 2213-2232 3′ UTR 31786GGTGGCAGATGACTGTAGGC 255 2218-2237 3′ UTR 31626 AGGTGTGGTGGCAGATGACT 212224-2243 3′ UTR 31470 AATTAGCCAGGTGTGGTGGC 256 2232-2251 3′ UTR 31627GTCTCTACTAAAAGTACAAA 257 2253-2272 3′ UTR 31628 CGGTGAAACCCTGTCTCTAC 2582265-2284 3′ UTR 31787 TGGCTAACACGGTGAAACCC 259 2274-2293 3′ UTR 31471AGACCATCCTGGCTAACACG 260 2283-2302 3′ UTR 31788 GAGATCGAGACCATCCTGGC 2612290-2309 3′ UTR 31629 GAGGTCAGGAGATCGAGACC 262 2298-2317 3′ UTR 31789GCGGATCACGAGGTCAGGAG 263 2307-2326 3′ UTR 31472 AGGCCGAGGTGGGCGGATCA 2642319-2338 3′ UTR 31790 TTTGGGAGGCCGAGGTGGGC 265 2325-2344 3′ UTR 31630TCCCAGCACTTTGGGAGGCC 266 2334-2353 3′ UTR 31791 CCTGTAATCCCAGCACTTTG 2672341-2360 3′ UTR 31631 GTGGCTCATGCCTGTAATCC 268 2351-2370 3′ UTR

[0104] TABLE 11 Nucleotide Sequences of Human mdm2 Chimeric (deoxygapped) Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ID NUCLEOTIDE TARGET NO. (5′ ->3′) NO: CO-ORDINATES² REGION 31393CAGCCAAGCTCGCGCGGTGC 3 0001-0020 5' UTR 31712 AAGCAGCCAAGCTCGCGCGG 330004-0023 5' UTR 31552 CAGGCCCCAGAAGCAGCCAA 34 0014-0033 5' UTR 31713GCCACACAGGCCCCAGAAGC 35 0020-0039 5' UTR 31394 ACACACAGGGCCACACAGGC 360029-0048 5' UTR 31714 TTCCGACACACAGGGCCACA 37 0034-0053 5' UTR 31553GCTCCATCTTTCCGACACAC 38 0043-0062 5' UTR 31715 GCTTCTTGCTCCATCTTTCC 390050-0069 5' UTR 31395 CCCTCGGGCTCGGCTTCTTG 40 0062-0081 5' UTR 31716GCGGCCGCCCCTCGGGCTCG 41 0070-0089 5' UTR 31554 AAGCAGCAGGATCTCGGTCA 420098-0107 5' UTR 31717 GCTGCGAAAGCAGCAGGATC 43 0105-0124 5' UTR 31396TGCTCCTGGCTGCGAAAGCA 44 0113-0132 5' UTR 31718 GGGACGGTGCTCCTGGCTGC 450120-0139 5' UTR 31555 ACTGGGCGCTCGTACGCACT 46 0150-0169 5' UTR 31719GCCAGGGCACTGGGCGCTCG 47 0158-0177 5' UTR 31397 TCTCCGGGCCAGGGCACTGG 480165-0184 5' UTR 31720 TCATTCCACTCTCCGGGCCA 49 0174-0193 5' UTR 31556GGAAGCACGACGCCCTGGGC 50 0202-0221 5' UTR 31721 TACTGCGGAAGCACGACGCC 510208-0227 5' UTR 31398 GGGACTGACTACTGCGGAAG 52 0217-0236 5' UTR 31722TCAAGACTCCCCAGTTTCCT 53 0242-0261 5' UTR 31557 CCTGCTCCTCACCATCCGGG 540289-0308 5' UTR 31399 TTTGCCTGCTCCTCACCATC 55 0293-0312 AUG 31400ATTTGCCTGCTCCTCACCAT 56 0294-0313 AUG 31401 CATTTGCCTGCTCCTCACCA 90295-0314 AUG 31402 ACATTTGCCTGCTCCTCACC 57 0296-0315 AUG 31403CACATTTGCCTGCTCCTCAC 58 0297-0316 AUG 31404 GCACATTTGCCTGCTCCTCA 590298-0317 AUG 31405 TGCACATTTGCCTGCTCCTC 60 0299-0318 AUG 31406TTGCACATTTGCCTGCTCCT 61 0300-0319 AUG 31407 ATTGCACATTTGCCTGCTCC 620301-0320 AUG 31408 TATTGCACATTTGCCTGCTC 63 0302-0321 AUG 31409GTATTGCACATTTCCCTGCT 10 0303-0322 AUG 31410 GGTATTGCACATTTGCCTGC 640304-0323 AUG 31411 TGGTATTGCACATTTGCCTG 65 0305-0324 AUG 31412TTGGTATTGCACATTTGCCT 66 0306-0325 AUG 31413 GTTGGTATTGCACATTTGCC 670307-0326 AUG 31414 TGTTGGTATTGCACATTTGC 68 0308-0327 AUG 31415ATGTTGGTATTGCACATTTG 69 0309-0328 AUG 31416 CATGTTGGTATTGCACATTT 700310-0329 AUG 31417 ACATGTTGGTATTGCACATT 71 0311-0330 AUG 31418GACATGTTGGTATTGCACAT 72 0312-0331 AUG 31419 AGACATGTTGGTATTGCACA 730313-0332 AUG 31420 CAGACATGTTGGTATTGCAC 74 0314-0333 AUG 31558CAGTAGGTACAGACATGTTG 75 0323-0342 coding 31723 TACAGCACCATCAGTAGGTA 760334-0353 coding 31421 GGAATCTGTGAGGTGGTTAC 77 0351-0370 coding 31559TTCCGAAGCTGGAATCTGTG 78 0361-0380 coding 31724 AGGGTCTCTTGTTCCGAAGC 790372-0391 coding 31422 GCTTTGGTCTAACCAGGGTC 80 0386-0405 coding 31560GCAATGGCTTTGGTCTAACC 81 0392-0411 coding 31725 TAACTTCAAAAGCAATGGCT 820403-0422 coding 31423 GTGCACCAACAGACTTTAAT 83 0422-0441 coding 31561ACCTCTTTCATAGTATAAGT 84 0450-0469 coding 31726 ATAATATACTGGCCAAGATA 850477-0496 coding 31424 TAATCGTTTAGTCATAATAT 86 0490-0509 coding 31727ATCATATAATCGTTTAGTCA 87 0496-0515 coding 31562 GCTTCTCATCATATAATCGT 880503-0522 coding 31728 CAATATGTTGTTGCTTCTCA 89 0515-0534 coding 31425GAACAATATACAATATGTTG 90 0525-0544 coding 31729 TCATTTGAACAATATACAAT 910531-0550 coding 31563 TAGAAGATCATTTGAACAAT 92 0538-0557 coding 31730AACAAATCTCCTAGAAGATC 93 0549-0568 coding 31426 TGGCACGCCAAACAAATCTC 940559-0578 coding 31731 AGAAGCTTGGCACGCCAAAC 95 0566-0585 coding 31564CTTTCACAGAGAAGCTTGGC 96 0575-0594 coding 31732 TTTTCCTGTGCTCTTTCACA 970587-0606 coding 31427 TATATATTTTCCTGTGCTCT 98 0593-0612 coding 31733ATCATGGTATATATTTTCCT 99 0600-0619 coding 31565 TTCCTGTAGATCATGGTATA 1000609-0628 coding 31734 TACTACCAAGTTCCTGTAGA 101 0619-0638 coding 31428TTCCTGCTGATTGACTACTA 102 0634-0653 coding 31566 TGAGTCCGATGATTCCTGCT 1030646-0665 coding 31735 CAGATGTACCTGAGTCCGAT 104 0656-0675 coding 31429CTGTTCTCACTCACAGATGT 105 0669-0688 coding 31567 TTCAAGGTGACACCTGTTCT 1060682-0701 coding 31736 ACTCCCACCTTCAAGGTGAC 107 0691-0710 coding 31430GGTCCTTTTGATCACTCCCA 108 0704-0723 coding 31568 AAGCTCTTGTACAAGGTCCT 1090718-0737 coding 31737 CTCTTCCTGAAGCTCTTGTA 110 0727-0746 coding 31431AAGATGAAGGTTTCTCTTCC 111 0740-0759 coding 31569 AAACCAAATGTGAAGATGAA 1120752-0771 coding 31738 ATGGTCTAGAAACCAAATGT 113 0761-0780 coding 31432CTAGATGAGGTAGATGGTCT 114 0774-0793 coding 31570 AATTGCTCTCCTTCTAGATG 1150787-0806 coding 31739 TCTGTCTCACTAATTGCTCT 116 0798-0817 coding 31433TCTGAATTTTCTTCTGTCTC 117 0810-0829 coding 31571 CACCAGATAATTCATCTGAA 1180824-0843 coding 31740 TTTGTCGTTCACCAGATAAT 119 0833-0852 coding 31434GTGGCGTTTTCTTTGTCGTT 120 0844-0863 coding 31572 TACTATCAGATTTGTGGCGT 1210857-0876 coding 31741 GAAAGGGAAATACTATCAGA 122 0867-0886 coding 31435GCTTTCATCAAAGGAAAGGG 123 0880-0899 coding 31573 TACACACAGAGCCAGGCTTT 1240895-0914 coding 31742 CTCCCTTATTACACACAGAG 125 0904-0923 coding 31436TCACAACATATCTCCCTTAT 126 0915-0934 coding 31574 CTACTGCTTCTTTCACAACA 1270927-0946 coding 31743 GATTCACTGCTACTGCTTCT 128 0936-0955 coding 31437TGGCGTCCCTGTAGATTCAC 129 0949-0968 coding 31575 AAGATCCGGATTCGATGGCG 1300964-0983 coding 31744 CAGCATCAAGATCCGGATTC 131 0971-0990 coding 31438GTTCACTTACACCAGCATCA 132 0983-1002 coding 31576 CAATCACCTGAATGTTCACT 1330996-1015 coding 31745 CTGATCCAACCAATCACCTG 134 1006-1025 coding 31439GAAACTGAATCCTGATCCAA 135 1017-1036 coding 31746 TGATCTGAAACTGAATCCTG 1361023-1042 coding 31577 CTACACTAAACTGATCTGAA 137 1034-1053 coding 31747CAACTTCAAATTCTACACTA 138 1046-1065 coding 31440 AGATTCAACTTCAAATTCTA 1391051-1070 coding 31748 GAGTCGAGAGATTCAACTTC 140 1059-1078 coding 31578TAATCTTCTGAGTCGAGAGA 141 1068-1087 coding 31749 CTAAGGCTATAATCTTCTGA 1421077-1096 coding 31441 TTCTTCACTAAGGCTATAAT 143 1084-1103 coding 31750TCTTGTCCTTCTTCACTAAG 144 1092-1111 coding 31579 CTGAGAGTTCTTGTCCTTCT 1451100-1119 coding 31751 TTCATCTGAGAGTTCTTGTC 146 1105-1124 coding 31442CCTCATCATCTTCATCTGAG 147 1115-1134 coding 31752 CTTGATATACCTCATCATCT 1481124-1143 coding 31753 ATACACAGTAACTTGATATA 149 1135-1154 coding 31443CTCTCCCCTGCCTGATACAC 150 1149-1168 coding 31580 GAATCTGTATCACTCTCCCC 1511161-1180 coding 31754 TCTTCAAATGAATCTGTATC 152 1170-1189 coding 31444AAATTTCAGGATCTTCTTCA 153 1184-1203 coding 31581 AGTCAGCTAAGGAAATTTCA 1541196-1215 coding 31755 GCATTTCCAATAGTCAGCTA 155 1207-1226 coding 31445CATTGCATGAAGTGCATTTC 156 1220-1239 coding 31756 TCATTTCATTGCATGAAGTG 1571226-1245 coding 31582 CATCTGTTGCAATGTGATGG 158 1257-1276 coding 31757GAAGGGCCCAACATCTGTTG 159 1268-1287 coding 31446 TTCTCACGAAGGGCCCAACA 1601275-1294 coding 31758 GAAGCCAATTCTCACGAACG 161 1283-1302 coding 31583TATCTTCAGGAAGCCAATTC 162 1292-1311 coding 31759 CTTTCCCTTTATCTTCAGGA 1631301-1320 coding 31447 TCCCCTTTATCTTTCCCTTT 164 1311-1330 coding 31584CTTTCTCAGAGATTTCCCCT 165 1325-1344 coding 31760 CAGTTTGGCTTTCTCAGAGA 1661333-1352 coding 31448 GTGTTGAGTTTTCCAGTTTG 167 1346-1365 coding 31585CCTCTTCAGCTTGTGTTGAG 168 1358-1377 coding 31761 ACATCAAAGCCCTCTTCAGC 1691368-1787 coding 31449 GAATCATTCACTATAGTTTT 170 1401-1420 coding 31586ATGACTCTCTGGAATCATTC 171 1412-1431 coding 31762 CCTCAACACATGACTCTCTG 1721421-1440 coding 31450 TTATCATCATTTTCCTCAAC 173 1434-1453 coding 31763TAATTTTATCATCATTTTCC 174 1439-1458 coding 31587 GAAGCTTGTGTAATTTTATC 1751449-1468 coding 31764 TGATTGTGAAGCTTGTGTAA 176 1456-1475 coding 31451CACTTTCTTGTGATTGTGAA 177 1466-1485 coding 31588 GCTGAGAATAGTCTTCACTT 1781481-1500 coding 31765 AGTTGATGGCTGAGAATAGT 179 1489-1508 coding 31452TGCTACTAGAAGTTGATGGC 180 1499-1518 coding 31766 TAAATAATGCTACTAGAAGT 1811506-1525 coding 31589 CTTGGCTGCTATAAATAATG 182 1517-1536 coding 31590ATCTTCTTGGCTGCTATAAA 183 1522-1541 coding 31453 AACTCTTTCACATCTTCTTG 1841533-1552 coding 31767 CCCTTTCAAACTCTTTCACA 185 1541-1560 coding 31591GGGTTTCTTCCCTTTCAAAC 186 1550-1569 coding 31768 TCTTTGTCTTGGGTTTCTTC 1871560-1579 coding 31454 CTCTCTTCTTTGTCTTGGGT 188 1566-1585 coding 31592AACTAGATTCCACACTCTCT 189 1580-1599 coding 31769 CAAGGTTCAATGGCATTAAG 1901605-1624 coding 31455 TGACAAATCACACAAGGTTC 191 1617-1636 coding 31593TCGACCTTGACAZATCACAC 192 1624-1643 coding 31594 ATGGACAATGCAACCATTTT 1931648-1667 coding 31770 TGTTTTGCCATGGACAATGC 194 1657-1676 coding 31456TAAGATGTCCTGTTTTGCCA 195 1667-1686 coding 31595 GCAGGCCATAAGATGTCCTG 1961675-1694 coding 31596 ACATGTAAAGCAGGCCATAA 197 1684-1703 coding 31771CTTTGCACATGTAAAGCAGG 198 1690-1709 coding 31457 TTTCTTTAGCTTCTTTGCAC 1991702-1721 coding 31597 TTATTCCTTTTCTTTAGCTT 200 1710-1729 coding 31598TGGGCAGGGCTTATTCCTTT 201 1720-1739 coding 31772 ACATACTGGGCAGGGCTTAT 2021726-1745 coding 31458 TTGGTTGTCTACATACTGGG 203 1736-1755 coding 31599TCATTTGAATTGGTTGTCTA 204 1745-1764 coding 31600 AAGTTAGCACAATCATTTGA 2051757-1776 coding 31601 TCTCTTATAGACAGGTCAAC 206 1787-1806 STOP 31459AAATATATAATTCTCTTATA 207 1798-1817 3' UTR 31602 AGTTAGAAATATATAATTCT 2081804-1823 3' UTR 31773 ATATAGTTAGAAATATATAA 209 1808-1827 3' UTR 31603CTAGGGTTATATAGTTAGAA 210 1816-1835 3' UTR 31774 TAAATTCCTAGGGTTATATA 2111823-1842 3' UTR 31460 CAGGTTGTCTJAATTCCTAG 212 1832-1851 3' UTR 31604ATAAATTTCAGGTTGTCTAA 213 1840-1859 3' UTR 31605 ATATATGTGAATAAATTTCA 2141850-1869 3' UTR 31606 CTTTGATATATGTGAATAAA 215 1855-1874 3' UTR 31461CATTTTCTCACTTTGATATA 216 1865-1884 3' UTR 31607 ATTGAGGCATTTTCTCACTT 2171872-1891 3' UTR 31608 AATCTATGTGAATTGAGGCA 218 1883-1902 3' UTR 31609AGAAGAAATCTATGTGAATT 219 1889-1908 3' UTR 31462 ATACTAAAGAGAAGAAATCT 2201898-1917 3' UTR 31610 GTCAATTATACTAAAGAGAA 221 1905-1924 3' UTR 31775TAGGTCAATTATACTAAAGA 222 1908-1927 3' UTR 31611 CAAAGTAGGTCAATTATACT 2231913-1932 3' UTR 31776 CCACTACCAAAGTAGGTCAA 224 1920-1939 3' UTR 31463AGTATTCACTATTCCACTAC 225 1933-1952 3' UTR 31612 TATAGTAAGTATTCACTATT 2261940-1959 3' UTR 31613 AGTCAAATTATAGTAAGTAT 227 1948-1967 3' UTR 31777CATATTCAAGTCAAATTATA 228 1956-1975 3' UTR 31464 AAAGGATGAGCTACATATTC 2291969-1988 3' UTR 31778 GTGTAAAGGATGAGCTACAT 230 1973-1992 3' UTR 31614TAGGAGTTGGTGTAAAGGAT 231 1982-2001 3' UTR 31779 TTTAAAATTAGGAGTTGGTG 2321990-2009 3' UTR 31615 GAAATTATTTAAAATTAGGA 233 1997-2016 3' UTR 31465CAGAGTAGAAATTATTTAAA 234 2004-2023 3' UTR 31616 CTCATTTAAGACAGAGTAGA 2352015-2034 3' UTR 31780 TACTTCTCATTTAXGACAGA 236 2020-2039 3' UTR 31617CATATACATATTTAAGAAAA 237 2051-2070 3' UTR 31466 TTAAATGTCATATACATATT 2382059-2078 3' UTR 31618 TAATAAGTTACATTTAAATG 239 2072-2091 3' UTR 31619GTAACAGAGCAAGACTCGGT 240 2103-2122 3' UTR 31467 CAGCCTGGGTPICAGAGCAA 2412111-2130 3' UTR 31781 CACTCCAGCCTGGGTAACAG 242 2116-2135 3' UTR 31620CCCACTGCACTCCAGCCTGG 243 2123-2142 3' UTR 31782 GCCAAGATCACCCACTGCAC 2442133-2152 3' UTR 31621 GCAGTGAGCCAAGATCACCC 245 2140-2159 3' UTR 31468GAGCTTGCAGTGAGCCAACA 246 2146-2165 3' UTR 31783 GAGGGCAGAGCTTGCAGTGA 2472153-2172 3' UTR 31622 CAGGAGAATGGTGCGAACCC 248 2176-2195 3' UTR 31623AGGCTGAGGCAGGAGAATGG 249 2185-2204 3' UTR 31784 ATTGGGAGGCTGAGGCAGGA 2502191-2210 3' UTR 31469 CAAGCTAATTGGGAGGCTGA 251 2198-2217 3' UTR 31624AGGCCAAGCTAATTGGGAGG 252 2202-2221 3' UTR 31785 ATGACTGTAGGCCAAGCTAA 2532210-2229 3' UTR 31625 CAGATGACTGTAGGCCAAGC 254 2213-2232 3' UTR 31786GGTGGCAGATGACTGTAGGC 255 2218-2237 3' UTR 31626 AGGTGTGGTGGCAGATGACT 212224-2243 3' UTR 31470 AATTAGCCAGGTGTGGTGGC 256 2232-2251 3' UTR 31627GTCTCTACTAAAAGTACAAA 257 2253-2272 3' UTR 31628 CGGTGAAACCCTGTCTCTAC 2582265-2284 3' UTR 31787 TGGCTAACACGGTGAAACCC 259 2274-2293 3' UTR 31471AGACCATCCTGGCTAACACG 260 2283-2302 3' UTR 31788 GAGATCGAGACCATCCTGGC 2612290-2309 3' UTR 31629 GAGGTCAGGAGATCGAGACC 262 2298-2317 3' UTR 31789GCGGATCACGAGGTCAGGAG 263 2307-2326 3' UTR 31472 AGGCCGAGGTGGGCGGATCA 2642319-2338 3' UTR 31790 TTTGGGAGGCCGAGGTGGGC 265 2325-2344 3' UTR 31630TCCCAGCACTTTGGGAGGCC 266 2334-2353 3' UTR 31791 CCTGTAATCCCAGCACTTTG 2672341-2360 3' UTR 31631 GTGGCTCATGCCTGTAATCC 268 2351-2370 3' UTR

[0105] Oligonucleotide activity was assayed by quantitation of mdm2 mRNAlevels by real-time PCR (RT-PCR) using the ABI PRISM™ 7700 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR, in which amplification products are quantitated after thePCR is completed, products in RT-PCR are quantitated as they accumulate.This is accomplished by including in the PCR reaction an oligonucleotideprobe that anneals specifically between the forward and reverse PCRprimers, and contains two fluorescent dyes. The primers and probes usedwere: Forward: 5′-GGCAAATGTGCAATACCAACA-3′ (SEQ ID NO. 269) Reverse:5′-TGCACCAACAGACTTTAATAACTTCA-3′(SEQ ID NO. 270) Probe:5′-FAM-CCACCTCACAGATTCCAGCTTCGGA-TAMRA-3′ (SEQ ID NO. 271)

[0106] A reporter dye (e.g., JOE or FAM, PE-Applied Biosystems, FosterCity, Calif.) was attached to the 5′ end of the probe and a quencher dye(e.g., TAMRA, PE-Applied Biosystems, Foster City, Calif.) was attachedto the 3′ end of the probe. When the probe and dyes are intact, reporterdye emission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the fluorescence intensity is monitored at regular(six-second) intervals by laser optics built into the ABI PRISM™ 7700Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

[0107] RT-PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μl PCRcocktail (1x TAQMAN® buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 U RNAse inhibitor, 1.25 units AMPLITAQ GOLD®, and 12.5 UMuLV reverse transcriptase) to 96 well plates containing 25 μl poly(A)mRNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD®, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0108] Results are shown in Table 12. Oligonucleotides 31394 (SEQ ID NO:36), 31398 (SEQ ID NO: 52), 31400 (SEQ ID NO: 56), 31402 (SEQ ID NO:57), 31405 (SEQ ID NO: 60), 31406 (SEQ ID NO: 61), 31415 (SEQ ID NO: 69), 31416 (SEQ ID NO: 70), 31418 (SEQ ID NO: 72) , 31434 (SEQ ID NO: 60),31436 (SEQ ID NO: 126), 31446 (SEQ ID NO: 160) , 31451 (SEQ ID NO: 177), 31452 (SEQ ID NO: 180), 31456 (SEQ ID NO: 195) , 31461 (SEQ ID NO:216) , 31468 (SEQ ID NO: 246) , 31469 (SEQ ID NO: 251) , 31471 (SEQ IDNO: 260) , and 31472 (SEQ ID NO: 264) gave at least approximately 50%reduction of mdm2 mRNA levels. TABLE 12 Activities of PhosphorothioateOligodeoxynucleotides Targeted to Human mdm2 SEQ GENE ISIS ID TARGET %mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION LIPOFECTIN^(T) — — 100%0% ^(M) only — 31393 3 5' UTR 59% 41% 31394 36 5' UTR 27% 73% 31395 405' UTR 96% 4% 31396 44 5' UTR 99% 1% 31397 48 5' UTR 76% 24% 31398 52 5'UTR 51% 49% 31399 55 AUG 138% — 31400 56 AUG 22% 78% 31401 9 AUG 69% 31%31402 57 AUG 47% 53% 31403 58 AUG 77% 23% 31404 59 AUG 60% 40% 31405 60AUG 35% 65% 31406 61 AUG 45% 55% 31407 62 AUG 65% 35% 31408 63 AUG 71%29% 31409 10 AUG 849% — 31410 64 AUG 79% 21% 31411 65 AUG 67% 33% 3141266 AUG 99% 1% 31413 67 AUG 68% 32% 31414 68 AUG 64% 36% 31415 69 AUG 48%52% 31416 70 AUG 36% 64% 31417 71 AUG 77% 23% 31418 72 AUG 53% 47% 3141973 AUG 122% — 31420 74 AUG 57% 43% 31421 77 coding 111% — 31422 80coding 85% 15% 31423 83 coding 126% — 31424 86 coding 70% 30% 31425 90coding 95% 5% 31426 94 coding 69% 31% 31427 98 coding 9465% — 31428 102coding 81% 19% 31429 105 coding 138% — 31430 108 coding 114% — 31431 111coding 77% 23% 31432 114 coding 676% — 31433 117 coding 145% — 31434 120coding 40% 60% 31435 123 coding 193% — 31436 126 coding 49% 51% 31437129 coding 146% — 31438 132 coding 76% 24% 31439 135 coding 104% — 31440139 coding 95% 5% 31441 143 coding 324% — 31442 147 coding 1840% — 31443150 coding 369% — 31444 153 coding 193% — 31445 156 coding 106% — 31446160 coding 29% 71% 31447 164 coding 82% 18% 31448 167 coding 117% —31449 170 coding 1769% — 31450 173 coding 84% 16% 31451 177 coding 49%51% 31452 180 coding 33% 67% 31453 184 coding 59% 41% 31454 188 coding171% — 31455 191 coding 61% 39% 31456 195 coding 42% 58% 31457 199coding 70% 30% 31458 203 coding 60% 40% 31459 207 3' UTR 149% — 31460212 3' UTR 71% 29% 31461 216 3' UTR 52% 48% 31462 220 3' UTR 1113% —31463 225 3' UTR 78% 22% 31464 229 3' UTR 112% — 31465 234 3' UTR 66%34% 31466 238 3' UTR 212% — 31467 241 3' UTR 77% 23% 31468 246 3' UTR17% 83% 31469 251 3' UTR 36% 64% 31470 256 3' UTR 60% 40% 31471 260 3'UTR 43% 57% 31472 264 3' UTR 35% 65%

Example 10 Effect of mdm2 Antisense Oligonucleotides on the Growth ofHuman A549 Lung Tumor Cells in Nude Mice

[0109] 200 μl of A549 cells (5×10⁶ cells) are implanted subcutaneouslyin the inner thigh of nude mice. mdm2 antisense oligonucleotides areadministered twice weekly for four weeks, beginning one week followingtumor cell inoculation. Oligonucleotides are formulated with cationiclipids (LIPOFECTIN™L) and given subcutaneously in the vicinity of thetumor. Oligonucleotide dosage was 5 mg/kg with 60 mg/kg cationic lipid.Tumor size is recorded weekly.

[0110] Activity of the oligonucleotides is measured by reduction intumor size compared to controls.

Example 11 U-87 Human Glioblastoma Cell Culture and SubcutaneousXenografts into Nude Mice

[0111] The U-87 human glioblastoma cell line is obtained from the ATCC(Manassas, Va.) and maintained in Iscove's DMEM medium supplemented withheat-inactivated 10% fetal calf serum (Yazaki, T., et al., Mol.Pharmacol., 1996, 50, 236-242). Nude mice are injected subcutaneouslywith 2× 10⁷ cells. Mice are injected intraperitoneally witholigonucleotide at dosages of either 2 mg/kg or 20 mg/kg for 21consecutive days beginning 7 days after xenografts were implanted. Tumorvolumes are measured on days 14, 21, 24, 31 and 35. Activity is measureby a reduced tumor volume compared to saline or sense oligonucleotidecontrols.

Example 12 Intracerebral U-87 Glioblastoma Xenografts into Nude Mice

[0112] U-87 cells are implanted in the brains of nude mice (Yazaki, T.,et al., Mol. Pharmacol., 1996, 50, 236-242). Mice are treated viacontinuous intraperitoneal administration of antisense oligonucleotide(20 mg/kg), control sense oligonucleotide (20 mg/kg) or saline beginningon day 7 after xenograft implantation. Activity of the oligonucleotideis measured by an increased survival time compared to controls.

1 271 2372 base pairs Nucleic Acid Single Unknown No not provided J.D.Kinzler,K.W. Meltzer,P.S. George,D.L. Vogelstein,B. Oliner Amplificationof a gene encoding a p53-associated protein in human sarcomas Nature 3586381 80-83 02-JUL-1992 1 GCACCGCGCG AGCTTGGCTG CTTCTGGGGC CTGTGTGGCCCTGTGTGTCG 50 GAAAGATGGA GCAAGAAGCC GAGCCCGAGG GGCGGCCGCG ACCCCTCTGA 100CCGAGATCCT GCTGCTTTCG CAGCCAGGAG CACCGTCCCT CCCCGGATTA 150 GTGCGTACGAGCGCCCAGTG CCCTGGCCCG GAGAGTGGAA TGATCCCCGA 200 GGCCCAGGGC GTCGTGCTTCCGCAGTAGTC AGTCCCCGTG AAGGAAACTG 250 GGGAGTCTTG AGGGACCCCC GACTCCAAGCGCGAAAACCC CGGATGGTGA 300 GGAGCAGGCA AATGTGCAAT ACCAACATGT CTGTACCTACTGATGGTGCT 350 GTAACCACCT CACAGATTCC AGCTTCGGAA CAAGAGACCC TGGTTAGACC400 AAAGCCATTG CTTTTGAAGT TATTAAAGTC TGTTGGTGCA CAAAAAGACA 450CTTATACTAT GAAAGAGGTT CTTTTTTATC TTGGCCAGTA TATTATGACT 500 AAACGATTATATGATGAGAA GCAACAACAT ATTGTATATT GTTCAAATGA 550 TCTTCTAGGA GATTTGTTTGGCGTGCCAAG CTTCTCTGTG AAAGAGCACA 600 GGAAAATATA TACCATGATC TACAGGAACTTGGTAGTAGT CAATCAGCAG 650 GAATCATCGG ACTCAGGTAC ATCTGTGAGT GAGAACAGGTGTCACCTTGA 700 AGGTGGGAGT GATCAAAAGG ACCTTGTACA AGAGCTTCAG GAAGAGAAAC750 CTTCATCTTC ACATTTGGTT TCTAGACCAT CTACCTCATC TAGAAGGAGA 800GCAATTAGTG AGACAGAAGA AAATTCAGAT GAATTATCTG GTGAACGACA 850 AAGAAAACGCCACAAATCTG ATAGTATTTC CCTTTCCTTT GATGAAAGCC 900 TGGCTCTGTG TGTAATAAGGGAGATATGTT GTGAAAGAAG CAGTAGCAGT 950 GAATCTACAG GGACGCCATC GAATCCGGATCTTGATGCTG GTGTAAGTGA 1000 ACATTCAGGT GATTGGTTGG ATCAGGATTC AGTTTCAGATCAGTTTAGTG 1050 TAGAATTTGA AGTTGAATCT CTCGACTCAG AAGATTATAG CCTTAGTGAA1100 GAAGGACAAG AACTCTCAGA TGAAGATGAT GAGGTATATC AAGTTACTGT 1150GTATCAGGCA GGGGAGAGTG ATACAGATTC ATTTGAAGAA GATCCTGAAA 1200 TTTCCTTAGCTGACTATTGG AAATGCACTT CATGCAATGA AATGAATCCC 1250 CCCCTTCCAT CACATTGCAACAGATGTTGG GCCCTTCGTG AGAATTGGCT 1300 TCCTGAAGAT AAAGGGAAAG ATAAAGGGGAAATCTCTGAG AAAGCCAAAC 1350 TGGAAAACTC AACACAAGCT GAAGAGGGCT TTGATGTTCCTGATTGTAAA 1400 AAAACTATAG TGAATGATTC CAGAGAGTCA TGTGTTGAGG AAAATGATGA1450 TAAAATTACA CAAGCTTCAC AATCACAAGA AAGTGAAGAC TATTCTCAGC 1500CATCAACTTC TAGTAGCATT ATTTATAGCA GCCAAGAAGA TGTGAAAGAG 1550 TTTGAAAGGGAAGAAACCCA AGACAAAGAA GAGAGTGTGG AATCTAGTTT 1600 GCCCCTTAAT GCCATTGAACCTTGTGTGAT TTGTCAAGGT CGACCTAAAA 1650 ATGGTTGCAT TGTCCATGGC AAAACAGGACATCTTATGGC CTGCTTTACA 1700 TGTGCAAAGA AGCTAAAGAA AAGGAATAAG CCCTGCCCAGTATGTAGACA 1750 ACCAATTCAA ATGATTGTGC TAACTTATTT CCCCTAGTTG ACCTGTCTAT1800 AAGAGAATTA TATATTTCTA ACTATATAAC CCTAGGAATT TAGACAACCT 1850GAAATTTATT CACATATATC AAAGTGAGAA AATGCCTCAA TTCACATAGA 1900 TTTCTTCTCTTTAGTATAAT TGACCTACTT TGGTAGTGGA ATAGTGAATA 1950 CTTACTATAA TTTGACTTGAATATGTAGCT CATCCTTTAC ACCAACTCCT 2000 AATTTTAAAT AATTTCTACT CTGTCTTAAATGAGAAGTAC TTGGTTTTTT 2050 TTTTCTTAAA TATGTATATG ACATTTAAAT GTAACTTATTATTTTTTTTG 2100 AGACCGAGTC TTGCTCTGTT ACCCAGGCTG GAGTGCAGTG GGTGATCTTG2150 GCTCACTGCA AGCTCTGCCC TCCCCGGGTT CGCACCATTC TCCTGCCTCA 2200GCCTCCCAAT TAGCTTGGCC TACAGTCATC TGCCACCACA CCTGGCTAAT 2250 TTTTTGTACTTTTAGTAGAG ACAGGGTTTC ACCGTGTTAG CCAGGATGGT 2300 CTCGATCTCC TGACCTCGTGATCCGCCCAC CTCGGCCTCC CAAAGTGCTG 2350 GGATTACAGG CATGAGCCAC CG 2372 500base pairs Nucleic Acid Single Unknown No not provided A. Flusberg, D.Haupt, Y. Barak, Y. Oren, M. Zauberman A functional p53-responsiveintronic promoter is contained within the human mdm2 gene Nucleic AcidsRes. 23 14 2584-2592 25-JUL-1995 2 GCTGCGGGC CCCTGCGGCG CGGGAGGTCCGGATGATCGC AGGTGCCTGT 50 CGGGTCACTA GTGTGAACGC TGCGCGTAGT CTGGGCGGGATTGGGCCGGT 100 TCAGTGGGCA GGTTGACTCA GCTTTTCCTC TTGAGCTGGT CAAGTTCAGA150 CACGTTCCGA AACTGCAGTA AAAGGAGTTA AGTCCTGACT TGTCTCCAGC 200TGGGGCTATT TAAACCATGC ATTTTCCCAG CTGTGTTCAG TGGCGATTGG 250 AGGGTAGACCTGTGGGCACG GACGCACGCC ACTTTTTCTC TGCTGATCCA 300 GGTAAGCACC GACTTGCTTGTAGCTTTAGT TTTAACTGTT GTTTATGTTC 350 TTTATATATG ATGTATTTTC CACAGATGTTTCATGATTTC CAGTTTTCAT 400 CGTGTCTTTT TTTTCCTTGT AGGCAAATGT GCAATACCAACATGTCTGTA 450 CCTACTGATG GGGCTGTAAC CACCCCACAG ATTCCAGCTT CGGAACAAGA500 20 base pairs Nucleic Acid Single Linear Yes not provided 3CAGCCAAGCT CGCGCGGTGC 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 4 TCTTTCCGAC ACACAGGGCC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 5 CAGCAGGATC TCGGTCAGAG 20 20 base pairsNucleic Acid Single Linear Yes not provided 6 GGGCGCTCGT ACGCACTAAT 2020 base pairs Nucleic Acid Single Linear Yes not provided 7 TCGGGGATCATTCCACTCTC 20 20 base pairs Nucleic Acid Single Linear Yes not provided8 CGGGGTTTTC GCGCTTGGAG 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 9 CATTTGCCTG CTCCTCACCA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 10 GTATTGCACA TTTGCCTGCT 20 20 base pairsNucleic Acid Single Linear Yes not provided 11 AGCACCATCA GTAGGTACAG 2020 base pairs Nucleic Acid Single Linear Yes not provided 12 CTACCAAGTTCCTGTAGATC 20 20 base pairs Nucleic Acid Single Linear Yes not provided13 TCAACTTCAA ATTCTACACT 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 14 TTTACAATCA GGAACATCAA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 15 AGCTTCTTTG CACATGTAAA 20 20 base pairsNucleic Acid Single Linear Yes not provided 16 CAGGTCAACT AGGGGAAATA 2020 base pairs Nucleic Acid Single Linear Yes not provided 17 TCTTATAGACAGGTCAACTA 20 20 base pairs Nucleic Acid Single Linear Yes not provided18 TCCTAGGGTT ATATAGTTAG 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 19 AAGTATTCAC TATTCCACTA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 20 CCAAGATCAC CCACTGCACT 20 20 base pairsNucleic Acid Single Linear Yes not provided 21 AGGTGTGGTG GCAGATGACT 2020 base pairs Nucleic Acid Single Linear Yes not provided 22 CCTGTCTCTACTAAAAGTAC 20 20 base pairs Nucleic Acid Single Linear No not provided23 ACAAGCCTTC GCTCTACCGG 20 20 base pairs Nucleic Acid Single Linear Nonot provided 24 TTCAGCGCAT TTGTACATAA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 25 TCTTTCCGAC ACACAGGGCC 20 20 base pairsNucleic Acid Single Linear No not provided 26 AGCTTCTTTA TACATGTAAA 2020 base pairs Nucleic Acid Single Linear No not provided 27 AGCTTCTTTACACATGTAAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided28 CTACCCTCCA ATCGCCACTG 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 29 GGTCTACCCT CCAATCGCCA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 30 CGTGCCCACA GGTCTACCCT 20 20 base pairsNucleic Acid Single Linear Yes not provided 31 AAGTGGCGTG CGTCCGTGCC 2020 base pairs Nucleic Acid Single Linear Yes not provided 32 AAAGTGGCGTGCGTCCGTGC 20 20 base pairs Nucleic Acid Single Linear Yes not provided33 AAGCAGCCAA GCTCGCGCGG 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 34 CAGGCCCCAG AAGCAGCCAA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 35 GCCACACAGG CCCCAGAAGC 20 20 base pairsNucleic Acid Single Linear Yes not provided 36 ACACACAGGG CCACACAGGC 2020 base pairs Nucleic Acid Single Linear Yes not provided 37 TTCCGACACACAGGGCCACA 20 20 base pairs Nucleic Acid Single Linear Yes not provided38 GCTCCATCTT TCCGACACAC 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 39 GCTTCTTGCT CCATCTTTCC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 40 CCCTCGGGCT CGGCTTCTTG 20 20 base pairsNucleic Acid Single Linear Yes not provided 41 GCGGCCGCCC CTCGGGCTCG 2020 base pairs Nucleic Acid Single Linear Yes not provided 42 AAGCAGCAGGATCTCGGTCA 20 20 base pairs Nucleic Acid Single Linear Yes not provided43 GCTGCGAAAG CAGCAGGATC 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 44 TGCTCCTGGC TGCGAAAGCA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 45 GGGACGGTGC TCCTGGCTGC 20 20 base pairsNucleic Acid Single Linear Yes not provided 46 ACTGGGCGCT CGTACGCACT 2020 base pairs Nucleic Acid Single Linear Yes not provided 47 GCCAGGGCACTGGGCGCTCG 20 20 base pairs Nucleic Acid Single Linear Yes not provided48 TCTCCGGGCC AGGGCACTGG 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 49 TCATTCCACT CTCCGGGCCA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 50 GGAAGCACGA CGCCCTGGGC 20 20 base pairsNucleic Acid Single Linear Yes not provided 51 TACTGCGGAA GCACGACGCC 2020 base pairs Nucleic Acid Single Linear Yes not provided 52 GGGACTGACTACTGCGGAAG 20 20 base pairs Nucleic Acid Single Linear Yes not provided53 TCAAGACTCC CCAGTTTCCT 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 54 CCTGCTCCTC ACCATCCGGG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 55 TTTGCCTGCT CCTCACCATC 20 20 base pairsNucleic Acid Single Linear Yes not provided 56 ATTTGCCTGC TCCTCACCAT 2020 base pairs Nucleic Acid Single Linear Yes not provided 57 ACATTTGCCTGCTCCTCACC 20 20 base pairs Nucleic Acid Single Linear Yes not provided58 CACATTTGCC TGCTCCTCAC 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 59 GCACATTTGC CTGCTCCTCA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 60 TGCACATTTG CCTGCTCCTC 20 20 base pairsNucleic Acid Single Linear Yes not provided 61 TTGCACATTT GCCTGCTCCT 2020 base pairs Nucleic Acid Single Linear Yes not provided 62 ATTGCACATTTGCCTGCTCC 20 20 base pairs Nucleic Acid Single Linear Yes not provided63 TATTGCACAT TTGCCTGCTC 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 64 GGTATTGCAC ATTTGCCTGC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 65 TGGTATTGCA CATTTGCCTG 20 20 base pairsNucleic Acid Single Linear Yes not provided 66 TTGGTATTGC ACATTTGCCT 2020 base pairs Nucleic Acid Single Linear Yes not provided 67 GTTGGTATTGCACATTTGCC 20 20 base pairs Nucleic Acid Single Linear Yes not provided68 TGTTGGTATT GCACATTTGC 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 69 ATGTTGGTAT TGCACATTTG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 70 CATGTTGGTA TTGCACATTT 20 20 base pairsNucleic Acid Single Linear Yes not provided 71 ACATGTTGGT ATTGCACATT 2020 base pairs Nucleic Acid Single Linear Yes not provided 72 GACATGTTGGTATTGCACAT 20 20 base pairs Nucleic Acid Single Linear Yes not provided73 AGACATGTTG GTATTGCACA 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 74 CAGACATGTT GGTATTGCAC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 75 CAGTAGGTAC AGACATGTTG 20 20 base pairsNucleic Acid Single Linear Yes not provided 76 TACAGCACCA TCAGTAGGTA 2020 base pairs Nucleic Acid Single Linear Yes not provided 77 GGAATCTGTGAGGTGGTTAC 20 20 base pairs Nucleic Acid Single Linear Yes not provided78 TTCCGAAGCT GGAATCTGTG 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 79 AGGGTCTCTT GTTCCGAAGC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 80 GCTTTGGTCT AACCAGGGTC 20 20 base pairsNucleic Acid Single Linear Yes not provided 81 GCAATGGCTT TGGTCTAACC 2020 base pairs Nucleic Acid Single Linear Yes not provided 82 TAACTTCAAAAGCAATGGCT 20 20 base pairs Nucleic Acid Single Linear Yes not provided83 GTGCACCAAC AGACTTTAAT 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 84 ACCTCTTTCA TAGTATAAGT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 85 ATAATATACT GGCCAAGATA 20 20 base pairsNucleic Acid Single Linear Yes not provided 86 TAATCGTTTA GTCATAATAT 2020 base pairs Nucleic Acid Single Linear Yes not provided 87 ATCATATAATCGTTTAGTCA 20 20 base pairs Nucleic Acid Single Linear Yes not provided88 GCTTCTCATC ATATAATCGT 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 89 CAATATGTTG TTGCTTCTCA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 90 GAACAATATA CAATATGTTG 20 20 base pairsNucleic Acid Single Linear Yes not provided 91 TCATTTGAAC AATATACAAT 2020 base pairs Nucleic Acid Single Linear Yes not provided 92 TAGAAGATCATTTGAACAAT 20 20 base pairs Nucleic Acid Single Linear Yes not provided93 AACAAATCTC CTAGAAGATC 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 94 TGGCACGCCA AACAAATCTC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 95 AGAAGCTTGG CACGCCAAAC 20 20 base pairsNucleic Acid Single Linear Yes not provided 96 CTTTCACAGA GAAGCTTGGC 2020 base pairs Nucleic Acid Single Linear Yes not provided 97 TTTTCCTGTGCTCTTTCACA 20 20 base pairs Nucleic Acid Single Linear Yes not provided98 TATATATTTT CCTGTGCTCT 20 20 base pairs Nucleic Acid Single Linear Yesnot provided 99 ATCATGGTAT ATATTTTCCT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 100 TTCCTGTAGA TCATGGTATA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 101 TACTACCAAGTTCCTGTAGA 20 20 base pairs Nucleic Acid Single Linear Yes not provided102 TTCCTGCTGA TTGACTACTA 20 20 base pairs Nucleic Acid Single LinearYes not provided 103 TGAGTCCGAT GATTCCTGCT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 104 CAGATGTACC TGAGTCCGAT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 105 CTGTTCTCACTCACAGATGT 20 20 base pairs Nucleic Acid Single Linear Yes not provided106 TTCAAGGTGA CACCTGTTCT 20 20 base pairs Nucleic Acid Single LinearYes not provided 107 ACTCCCACCT TCAAGGTGAC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 108 GGTCCTTTTG ATCACTCCCA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 109 AAGCTCTTGTACAAGGTCCT 20 20 base pairs Nucleic Acid Single Linear Yes not provided110 CTCTTCCTGA AGCTCTTGTA 20 20 base pairs Nucleic Acid Single LinearYes not provided 111 AAGATGAAGG TTTCTCTTCC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 112 AAACCAAATG TGAAGATGAA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 113 ATGGTCTAGAAACCAAATGT 20 20 base pairs Nucleic Acid Single Linear Yes not provided114 CTAGATGAGG TAGATGGTCT 20 20 base pairs Nucleic Acid Single LinearYes not provided 115 AATTGCTCTC CTTCTAGATG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 116 TCTGTCTCAC TAATTGCTCT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 117 TCTGAATTTTCTTCTGTCTC 20 20 base pairs Nucleic Acid Single Linear Yes not provided118 CACCAGATAA TTCATCTGAA 20 20 base pairs Nucleic Acid Single LinearYes not provided 119 TTTGTCGTTC ACCAGATAAT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 120 GTGGCGTTTT CTTTGTCGTT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 121 TACTATCAGATTTGTGGCGT 20 20 base pairs Nucleic Acid Single Linear Yes not provided122 GAAAGGGAAA TACTATCAGA 20 20 base pairs Nucleic Acid Single LinearYes not provided 123 GCTTTCATCA AAGGAAAGGG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 124 TACACACAGA GCCAGGCTTT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 125 CTCCCTTATTACACACAGAG 20 20 base pairs Nucleic Acid Single Linear Yes not provided126 TCACAACATA TCTCCCTTAT 20 20 base pairs Nucleic Acid Single LinearYes not provided 127 CTACTGCTTC TTTCACAACA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 128 GATTCACTGC TACTGCTTCT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 129 TGGCGTCCCTGTAGATTCAC 20 20 base pairs Nucleic Acid Single Linear Yes not provided130 AAGATCCGGA TTCGATGGCG 20 20 base pairs Nucleic Acid Single LinearYes not provided 131 CAGCATCAAG ATCCGGATTC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 132 GTTCACTTAC ACCAGCATCA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 133 CAATCACCTGAATGTTCACT 20 20 base pairs Nucleic Acid Single Linear Yes not provided134 CTGATCCAAC CAATCACCTG 20 20 base pairs Nucleic Acid Single LinearYes not provided 135 GAAACTGAAT CCTGATCCAA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 136 TGATCTGAAA CTGAATCCTG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 137 CTACACTAAACTGATCTGAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided138 CAACTTCAAA TTCTACACTA 20 20 base pairs Nucleic Acid Single LinearYes not provided 139 AGATTCAACT TCAAATTCTA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 140 GAGTCGAGAG ATTCAACTTC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 141 TAATCTTCTGAGTCGAGAGA 20 20 base pairs Nucleic Acid Single Linear Yes not provided142 CTAAGGCTAT AATCTTCTGA 20 20 base pairs Nucleic Acid Single LinearYes not provided 143 TTCTTCACTA AGGCTATAAT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 144 TCTTGTCCTT CTTCACTAAG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 145 CTGAGAGTTCTTGTCCTTCT 20 20 base pairs Nucleic Acid Single Linear Yes not provided146 TTCATCTGAG AGTTCTTGTC 20 20 base pairs Nucleic Acid Single LinearYes not provided 147 CCTCATCATC TTCATCTGAG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 148 CTTGATATAC CTCATCATCT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 149 ATACACAGTAACTTGATATA 20 20 base pairs Nucleic Acid Single Linear Yes not provided150 CTCTCCCCTG CCTGATACAC 20 20 base pairs Nucleic Acid Single LinearYes not provided 151 GAATCTGTAT CACTCTCCCC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 152 TCTTCAAATG AATCTGTATC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 153 AAATTTCAGGATCTTCTTCA 20 20 base pairs Nucleic Acid Single Linear Yes not provided154 AGTCAGCTAA GGAAATTTCA 20 20 base pairs Nucleic Acid Single LinearYes not provided 155 GCATTTCCAA TAGTCAGCTA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 156 CATTGCATGA AGTGCATTTC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 157 TCATTTCATTGCATGAAGTG 20 20 base pairs Nucleic Acid Single Linear Yes not provided158 CATCTGTTGC AATGTGATGG 20 20 base pairs Nucleic Acid Single LinearYes not provided 159 GAAGGGCCCA ACATCTGTTG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 160 TTCTCACGAA GGGCCCAACA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 161 GAAGCCAATTCTCACGAAGG 20 20 base pairs Nucleic Acid Single Linear Yes not provided162 TATCTTCAGG AAGCCAATTC 20 20 base pairs Nucleic Acid Single LinearYes not provided 163 CTTTCCCTTT ATCTTCAGGA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 164 TCCCCTTTAT CTTTCCCTTT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 165 CTTTCTCAGAGATTTCCCCT 20 20 base pairs Nucleic Acid Single Linear Yes not provided166 CAGTTTGGCT TTCTCAGAGA 20 20 base pairs Nucleic Acid Single LinearYes not provided 167 GTGTTGAGTT TTCCAGTTTG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 168 CCTCTTCAGC TTGTGTTGAG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 169 ACATCAAAGCCCTCTTCAGC 20 20 base pairs Nucleic Acid Single Linear Yes not provided170 GAATCATTCA CTATAGTTTT 20 20 base pairs Nucleic Acid Single LinearYes not provided 171 ATGACTCTCT GGAATCATTC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 172 CCTCAACACA TGACTCTCTG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 173 TTATCATCATTTTCCTCAAC 20 20 base pairs Nucleic Acid Single Linear Yes not provided174 TAATTTTATC ATCATTTTCC 20 20 base pairs Nucleic Acid Single LinearYes not provided 175 GAAGCTTGTG TAATTTTATC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 176 TGATTGTGAA GCTTGTGTAA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 177 CACTTTCTTGTGATTGTGAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided178 GCTGAGAATA GTCTTCACTT 20 20 base pairs Nucleic Acid Single LinearYes not provided 179 AGTTGATGGC TGAGAATAGT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 180 TGCTACTAGA AGTTGATGGC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 181 TAAATAATGCTACTAGAAGT 20 20 base pairs Nucleic Acid Single Linear Yes not provided182 CTTGGCTGCT ATAAATAATG 20 20 base pairs Nucleic Acid Single LinearYes not provided 183 ATCTTCTTGG CTGCTATAAA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 184 AACTCTTTCA CATCTTCTTG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 185 CCCTTTCAAACTCTTTCACA 20 20 base pairs Nucleic Acid Single Linear Yes not provided186 GGGTTTCTTC CCTTTCAAAC 20 20 base pairs Nucleic Acid Single LinearYes not provided 187 TCTTTGTCTT GGGTTTCTTC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 188 CTCTCTTCTT TGTCTTGGGT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 189 AACTAGATTCCACACTCTCT 20 20 base pairs Nucleic Acid Single Linear Yes not provided190 CAAGGTTCAA TGGCATTAAG 20 20 base pairs Nucleic Acid Single LinearYes not provided 191 TGACAAATCA CACAAGGTTC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 192 TCGACCTTGA CAAATCACAC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 193 ATGGACAATGCAACCATTTT 20 20 base pairs Nucleic Acid Single Linear Yes not provided194 TGTTTTGCCA TGGACAATGC 20 20 base pairs Nucleic Acid Single LinearYes not provided 195 TAAGATGTCC TGTTTTGCCA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 196 GCAGGCCATA AGATGTCCTG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 197 ACATGTAAAGCAGGCCATAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided198 CTTTGCACAT GTAAAGCAGG 20 20 base pairs Nucleic Acid Single LinearYes not provided 199 TTTCTTTAGC TTCTTTGCAC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 200 TTATTCCTTT TCTTTAGCTT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 201 TGGGCAGGGCTTATTCCTTT 20 20 base pairs Nucleic Acid Single Linear Yes not provided202 ACATACTGGG CAGGGCTTAT 20 20 base pairs Nucleic Acid Single LinearYes not provided 203 TTGGTTGTCT ACATACTGGG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 204 TCATTTGAAT TGGTTGTCTA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 205 AAGTTAGCACAATCATTTGA 20 20 base pairs Nucleic Acid Single Linear Yes not provided206 TCTCTTATAG ACAGGTCAAC 20 20 base pairs Nucleic Acid Single LinearYes not provided 207 AAATATATAA TTCTCTTATA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 208 AGTTAGAAAT ATATAATTCT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 209 ATATAGTTAGAAATATATAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided210 CTAGGGTTAT ATAGTTAGAA 20 20 base pairs Nucleic Acid Single LinearYes not provided 211 TAAATTCCTA GGGTTATATA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 212 CAGGTTGTCT AAATTCCTAG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 213 ATAAATTTCAGGTTGTCTAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided214 ATATATGTGA ATAAATTTCA 20 20 base pairs Nucleic Acid Single LinearYes not provided 215 CTTTGATATA TGTGAATAAA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 216 CATTTTCTCA CTTTGATATA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 217 ATTGAGGCATTTTCTCACTT 20 20 base pairs Nucleic Acid Single Linear Yes not provided218 AATCTATGTG AATTGAGGCA 20 20 base pairs Nucleic Acid Single LinearYes not provided 219 AGAAGAAATC TATGTGAATT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 220 ATACTAAAGA GAAGAAATCT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 221 GTCAATTATACTAAAGAGAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided222 TAGGTCAATT ATACTAAAGA 20 20 base pairs Nucleic Acid Single LinearYes not provided 223 CAAAGTAGGT CAATTATACT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 224 CCACTACCAA AGTAGGTCAA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 225 AGTATTCACTATTCCACTAC 20 20 base pairs Nucleic Acid Single Linear Yes not provided226 TATAGTAAGT ATTCACTATT 20 20 base pairs Nucleic Acid Single LinearYes not provided 227 AGTCAAATTA TAGTAAGTAT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 228 CATATTCAAG TCAAATTATA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 229 AAAGGATGAGCTACATATTC 20 20 base pairs Nucleic Acid Single Linear Yes not provided230 GTGTAAAGGA TGAGCTACAT 20 20 base pairs Nucleic Acid Single LinearYes not provided 231 TAGGAGTTGG TGTAAAGGAT 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 232 TTTAAAATTA GGAGTTGGTG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 233 GAAATTATTTAAAATTAGGA 20 20 base pairs Nucleic Acid Single Linear Yes not provided234 CAGAGTAGAA ATTATTTAAA 20 20 base pairs Nucleic Acid Single LinearYes not provided 235 CTCATTTAAG ACAGAGTAGA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 236 TACTTCTCAT TTAAGACAGA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 237 CATATACATATTTAAGAAAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided238 TTAAATGTCA TATACATATT 20 20 base pairs Nucleic Acid Single LinearYes not provided 239 TAATAAGTTA CATTTAAATG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 240 GTAACAGAGC AAGACTCGGT 20 20 basepairs Nucleic Acid Single Linear Yes not provided 241 CAGCCTGGGTAACAGAGCAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided242 CACTCCAGCC TGGGTAACAG 20 20 base pairs Nucleic Acid Single LinearYes not provided 243 CCCACTGCAC TCCAGCCTGG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 244 GCCAAGATCA CCCACTGCAC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 245 GCAGTGAGCCAAGATCACCC 20 20 base pairs Nucleic Acid Single Linear Yes not provided246 GAGCTTGCAG TGAGCCAAGA 20 20 base pairs Nucleic Acid Single LinearYes not provided 247 GAGGGCAGAG CTTGCAGTGA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 248 CAGGAGAATG GTGCGAACCC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 249 AGGCTGAGGCAGGAGAATGG 20 20 base pairs Nucleic Acid Single Linear Yes not provided250 ATTGGGAGGC TGAGGCAGGA 20 20 base pairs Nucleic Acid Single LinearYes not provided 251 CAAGCTAATT GGGAGGCTGA 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 252 AGGCCAAGCT AATTGGGAGG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 253 ATGACTGTAGGCCAAGCTAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided254 CAGATGACTG TAGGCCAAGC 20 20 base pairs Nucleic Acid Single LinearYes not provided 255 GGTGGCAGAT GACTGTAGGC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 256 AATTAGCCAG GTGTGGTGGC 20 20 basepairs Nucleic Acid Single Linear Yes not provided 257 GTCTCTACTAAAAGTACAAA 20 20 base pairs Nucleic Acid Single Linear Yes not provided258 CGGTGAAACC CTGTCTCTAC 20 20 base pairs Nucleic Acid Single LinearYes not provided 259 TGGCTAACAC GGTGAAACCC 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 260 AGACCATCCT GGCTAACACG 20 20 basepairs Nucleic Acid Single Linear Yes not provided 261 GAGATCGAGACCATCCTGGC 20 20 base pairs Nucleic Acid Single Linear Yes not provided262 GAGGTCAGGA GATCGAGACC 20 20 base pairs Nucleic Acid Single LinearYes not provided 263 GCGGATCACG AGGTCAGGAG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 264 AGGCCGAGGT GGGCGGATCA 20 20 basepairs Nucleic Acid Single Linear Yes not provided 265 TTTGGGAGGCCGAGGTGGGC 20 20 base pairs Nucleic Acid Single Linear Yes not provided266 TCCCAGCACT TTGGGAGGCC 20 20 base pairs Nucleic Acid Single LinearYes not provided 267 CCTGTAATCC CAGCACTTTG 20 20 base pairs Nucleic AcidSingle Linear Yes not provided 268 GTGGCTCATG CCTGTAATCC 20 21 basepairs Nucleic Acid Single Linear Yes not provided 269 GGCAAATGTGCAATACCAAC A 21 26 base pairs Nucleic Acid Single Linear Yes notprovided 270 TGCACCAACA GACTTTAATA ACTTCA 26 25 base pairs Nucleic AcidSingle Linear Yes not provided 271 CCACCTCACA GATTCCAGCT TCGGA 25

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases inlength targeted to the 5′ untranslated region, translation terminationcodon region or 3′ untranslated region of a nucleic acid moleculeencoding human mdm2, wherein said antisense compound modulates theexpression of human mdm2.
 2. The antisense compound of claim 1 whereinsaid antisense compound inhibits the expression of human mdm2.
 3. Theantisense compound of claim 1 which is an antisense oligonucleotide. 4.An antisense compound up to 30 nucleobases in length comprising at leastan 8-nucleobase portion of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 216, SEQ ID NO: 246, SEQ ID NO:251, SEQ ID NO: 260, or SEQ ID NO: 264 which inhibits the expression ofhuman mdm2.
 5. The antisense compound of claim 2 which is targeted tothe 5′ untranslated region of the S-mdm2 transcript.
 6. The antisensecompound of claim 5 comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5or SEQ ID NO:
 7. 7. The antisense compound of claim 2 comprising SEQ IDNO:
 4. 8. The antisense compound of claim 1 which contains at least onephosphorothioate intersugar linkage.
 9. The antisense compound of claim1 which has at least one 2′-O-methoxyethyl modification.
 10. Theantisense compound of claim 1 which contains at least one 5-methylcytidine.
 11. The antisense compound of claim 10 in which every2′-O-methoxyethyl modified cytidine residue is a 5-methyl cytidine. 12.A pharmaceutical composition comprising the antisense compound of claim1 and a pharmaceutically acceptable carrier or diluent.
 13. Thepharmaceutical composition of claim 12 wherein said pharmaceuticallyacceptable carrier or diluent further comprises a lipid or liposome. 14.A method of modulating the expression of human mdm2 in cells or tissuescomprising contacting said cells or tissues with the antisense compoundof claim 1 .
 15. A method of reducing hyperproliferation of human cellscomprising contacting proliferating human cells with the antisensecompound of claim 2 or a pharmaceutical composition comprising saidantisense compound.
 16. A method of treating an animal having a diseaseor condition associated with mdm2 comprising administering to saidanimal a therapeutically or prophylactically effective amount of anantisense compound of claim 1 .
 17. The method of claim 16 wherein thedisease or condition is associated with overexpression of mdm2 and theantisense compound inhibits the expression of mdm2.
 18. The method ofclaim 16 wherein the disease or condition is associated withamplification of the mdm2 gene and the antisense compound inhibits theexpression of mdm2.
 19. The method of claim 16 wherein the disease orcondition is a hyperproliferative condition and the antisense compoundinhibits the expression of mdm2.
 20. The method of claim 19 wherein thehyperproliferative condition is cancer.
 21. The method of claim 20wherein the cancer is a blood, brain, breast, lung or a soft tissuecancer.
 22. The method of claim 19 wherein the hyperproliferativecondition is psoriasis, fibrosis, atherosclerosis or restenosis.
 23. Themethod of claim 16 wherein said antisense compound is administered incombination with a chemotherapeutic agent to overcome drug resistance.24. An antisense compound up to 30 nucleobases in length targeted to thecoding region or translational start site of a nucleic acid moleculeencoding human mdm2, wherein said antisense compound inhibits theexpression of said human mdm2 and comprises at least an 8-nucleobaseportion of SEQ ID NO: 15, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:120, SEQ ID NO: 126, SEQ ID NO: 160, SEQ ID NO: 177, SEQ ID NO: 180, orSEQ ID NO:
 195. 25. The antisense compound of claim 24 which contains atleast one phosphorothioate intersugar linkage.
 26. The antisensecompound of claim 24 which has at least one 2′-O-methoxyethylmodification.
 27. The antisense compound of claim 24 which contains atleast one 5-methyl cytidine.
 28. The antisense compound of claim 27 inwhich every 2′-O-methoxyethyl modified cytidine residue is a 5-methylcytidine.
 29. A pharmaceutical composition comprising the antisensecompound of claim 24 and a pharmaceutically acceptable carrier ordiluent.
 30. The pharmaceutical composition of claim 29 wherein saidpharmaceutically acceptable carrier or diluent comprises a lipid orliposome.
 31. A method of modulating the expression of human mdm2 incells or tissues comprising contacting said cells or tissues with theantisense compound of claim 24 .
 32. A method of reducinghyperproliferation of human cells comprising contacting proliferatinghuman cells with the antisense compound of claim 24 .
 33. A method ofreducing hyperproliferation of human cells comprising contactingproliferating human cells with the pharmaceutical composition of claim29 .
 34. A method of treating an animal having a disease or conditionassociated with mdm2 comprising administering to said animal atherapeutically or prophylactically effective amount of the antisensecompound of claim 24 .
 35. The method of claim 34 wherein the disease orcondition is associated with overexpression of mdm2 and the antisensecompound inhibits the expression of mdm2.
 36. The method of claim 34wherein the disease or condition is associated with amplification of themdm2 gene and the antisense compound inhibits the expression of mdm2.37. The method of claim 34 wherein the disease or condition is ahyperproliferative condition and the antisense compound inhibits theexpression of mdm2.
 38. The method of claim 37 wherein thehyperproliferative condition is cancer.
 39. The method of claim 38wherein the cancer is a blood, brain, breast, lung or a soft tissuecancer.
 40. The method of claim 37 wherein the hyperproliferativecondition is psoriasis, fibrosis, atherosclerosis or restenosis.
 41. Themethod of claim 34 wherein said antisense compound is administered incombination with a chemotherapeutic agent to overcome drug resistance.